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Physio-biochemical mechanism of melatonin seed priming in stimulating growth and drought tolerance in bread wheat

BMC Plant Biology volume 24, Article number: 918 (2024) Cite this article

Abstract

Drought stress (DS) adversely affects a plant’s development and growth by negatively altering the plant’s physio-biochemical functions. Previous investigations have illustrated that seed priming with growth regulators is an accessible, affordable, and effective practice to elevate a plant’s tolerance to drought stress. Melatonin (MT) is derived from the precursor tryptophan and can improve germination, biomass, and photosynthesis under stress conditions. The current study examined the effect of melatonin seed priming on two wheat cultivars (Fakhar-e-Bhakkar and Akber-19) cultivated under severe drought conditions (35% FC). There were 6 levels of melatonin (i.e., M0 = control, M1 = 1 mg L− 1, M2 = 2 mg L− 1, M3 = 3 mg L− 1, M4 = 4 mg L− 1 and M5 = mg L− 1) which were used for seed priming. Our results confirmed that seed priming with M2 = 2 mgL− 1 concentration of MT alleviates the negative effects of DS by boosting the germination rate by 54.84% in Akber-19 and 33.33% in Fakhar-e-Bhakkar. Similarly, leaf-relative water contents were enhanced by 22.38% and 13.28% in Akber-19 and Fakhar-e-Bhakkar, respectively. Melatonin pre-treatment with 2 mgL− 1 significantly enhanced fresh and dry biomass of shoot and root, leaf area, photosynthetic pigments, osmoprotectants accumulation [total soluble proteins (TSP), total free amino acids (TFAA), proline, soluble sugars, glycine betaine (GB)] and lowered the amount of malondialdehyde (MDA) and hydrogen peroxide (H2O2) production by elevating antioxidants [Ascorbic acid, catalase (CAT), Phenolics, peroxidase (POD) and superoxide dismutase (SOD)] activity under drought stress (DS). Meanwhile, under control conditions (NoDS), the melatonin treatment M1 = 1 mgL− 1 effectively enhanced all the growth-related physio-biochemical attributes in both wheat cultivars. In the future, more investigations are suggested on different crops under variable agroclimatic conditions to declare 2 mgL− 1 melatonin as an efficacious amendment to alleviate drought stress.

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Introduction

Worldwide climatic alterations have enhanced the probability of short-term excessive climate stresses in food production, especially drought stress [1,2,3,4,5]. The destructive impacts of drought stress have made it challenging to achieve optimum crop production [6,7,8,9]. It can decrease germination, root proliferation, nutrient uptake, and synthesis of chlorophyll contents [10, 11]. Reduced water efficiency also limited leaf expansion and stem extension [12]. In addition, synthesizing reactive oxygen species (ROS) also causes oxidative stress, mitigated by plant antioxidant activity [13, 14]. On the other hand, damage caused by osmotic stress at the cellular level also resulted in poor crop productivity and growth [15,16,17].

Seed priming (SP) is an influential approach to improving abiotic stress tolerance in crops. It can notably boost germination and seedling growth in abiotic strain conditions [18,19,20,21]. It progresses seed emergence and seedling expansion, lessens crop growth period, and enhances yield output [22]. Seed priming maintains seed structure during imbibition, synthesizes RNA and proteins, has antioxidant mechanisms, and adjusts normal hydration levels [23]. However, using growth regulators for SP is an inexpensive, accessible, and effective practice to increase plant tolerance levels under stress environments. Various plant species covering main agronomic crops have been studied to analyze the impacts of seed priming. However exogenous melatonin application which can improve plant vigor under harmful environments [24] and increase stress tolerance still needs in depth investigations [25].

In plants, melatonin primarily acts as an antioxidant representative and regulates reactive oxygen species [26,27,28]. In addition, melatonin induces numerous particular physiological reactions in plants that could increase photosynthesis, root formation, carbon fixation, growth, and germination under abiotic stresses [29]. Frequently, melatonin is produced in mitochondria and chloroplast of leaves and roots and transported to meristem, fruits, and flowers in plants [30]. Melatonin is more efficient in scavenging ROS and regulating superoxide dismutase, peroxidase, and catalase enzymes. It reduces electron leakage by enhancing the electron transport chain efficacy [31]. However, various functional roles of melatonin as a regulatory agent in cereal crops like wheat are still under discussion [32].

Wheat (Triticum aestivum L.) is consumed globally as one of the leading cereal crops. Hence, variations in melatonin levels are a prominent factor in numerous drought-related wheat stress reactions [33, 34]. Previous research has found that exogenously applied melatonin could overcome oxidative injury due to drought stress in Zea mays [19], plus it enhanced seedling enhancement and seed germination in numerous crops [35].

Since wheat is a significant cereal, it is essential to enhance its metabolic adaptations, enabling it to thrive in drought conditions to achieve higher yields. Prior studies have demonstrated that seed priming with different growth regulators can enhance a plant’s ability to withstand drought conditions [1]. However, the investigations of melatonin seed priming in wheat crops and the selection of appropriate melatonin concentration still need more in-depth investigations. This study covers the knowledge gap regarding the best melatonin concentration as a seed priming agent against drought stress. It is hypothesized that a lower concentration of melatonin seed priming might potentially enhance wheat growth under drought stress.

Materials and methods

Experimental material and treatments

This research was established to evaluate the effects of melatonin seed priming with different concentrations (M0 = distilled water, M1 = 1mgL− 1, M2 = 2mgL− 1, M3 = 3mgL− 1, M4 = 4mgL− 1and M5 = 5mgL− 1), using 35 days old wheat plants under drought stress conditions. For this examination, a CRD factorial design was chosen with four replicates. Melatonin stock solution (5mgL− 1) was prepared, and further dilutions were made for the rest of the solutions. The seeds of 2 wheat cultivars, i.e., cv. Akber-19 and cv. Fakhar-e-Bhakkar was collected from Ayub Agriculture Research Institute (AARI), Faisalabad. Healthy seeds were sterilized and soaked in different melatonin concentrations and distilled water (M0-M5) for approximately 16 h before sowing. After soaking, the seeds were surface dried, and ten wheat seeds were planted individually in all plastic pots, having 5 kg loam soil. Drought stress was maintained (NoDS = control, DS = 35% FC) from the start of the experimentation. Field capacity was calculated according to the formula [36].

Field Capacity (FC) = Saturation percentage of sample/2.

Six plants were kept at the seedling stage after thinning in each pot. There were 2 levels of soil moisture were throughout the experiment, i.e., one was 100% FC labeled as control, while the second 35% and.

Soil preparation

The soil mix was prepared by mixing 3/4th loam and 1/4th organic compost. Thoroughly mixed, well-drained soil was then filled into pots. Before sowing, the soil was watered to field capacity and allowed to settle for 24 h. After sowing, the soil was maintained at a consistent moisture level throughout the experiment by watering as needed.

Fertilizer application

For the achievement of optimum crop growth, macronutrients nitrogen (N), phosphorus (P), and potassium (K) were applied at the rate of 120: 90 and 60 kg ha− 1, respectively [37]. Double application of a combination of NPK (20%) and trace elements (80%) was applied at 1 g/kg. One application before sowing and 2nd after 22 days. The nitrogen was applied in three split doses. Regarding P and K fertilizers, the recommended rates were applied in a single dose when sowing [37].

Seed germination percentage

Germination percentage was calculated by counting germinated seeds manually after 6 and 12 days of sowing in each treatment containing pot (4 replicates) by the formula:

Germination percentage = (Germinated seedlings/Total seeds sown) × 100.

Leaf relative water content determination

The relative water content (RWC) percentage of leaf samples was measured by the [38] method. The fresh weight (FW) of 2nd leaf was noted instantly after sampling on the 35th day of seed establishment. Then, the leaf sample was completely dipped in distilled water (DW) overnight, and turgid weight (TW) was noted for every sample. Afterward, the sample leaves were dehydrated in an oven at 72 °C for 2 days to assess dry weight (DW). Ultimately, the RWC percentage was computed by the following expression,

$$\:\text{R}\text{W}\text{C}\:\text{\%}=\frac{(\text{F}\text{W}-\text{D}\text{W})}{(\text{T}\text{W}-\text{D}\text{W})}\:\times\:100$$

Sample collection

After 35 days of germination, plant samples were uprooted. Four plant samples of identical size were taken from all pots for the morpho-physiological parameters and saved at low temperature (-20 °C) for biochemical attributes.

Morphological parameters determination

Fresh weights (g) of roots and shoots were noted when uprooting with electrical balance. Al-Karaki [39] method was applied to determine the dry weights of the root and shoot. After noting fresh weight, plant samples were packed carefully in paper packets and left in the oven for 48 h at 72 °C. After that, dry weights were noted carefully. A simple lab scale manually notes samples’ shoot and root lengths (cm). Leaf area (cm2) was counted manually by scale, leaf length and width (extreme) were noted, and the subsequent formulary was used to conclude leaf area per plant [40].

Leaf Area (cm2) = Leaf length × Leaf width (max.) × 0.75 (CF).

Photosynthetic pigments determination

Chlorophyll pigments (Chl. a, Chl. b, and carotenoids) were measured by the technique of [41]. Fresh leaf (0.1 g) was chopped into minute fragments and extracted with 5mL of 80% acetone for 24 h to analyze optical density at 663, 645, and 480 nm on a UV-VIS double beam spectrophotometer (Halo DB-20).

Biochemical Analysis

Preparation of enzyme extract for antioxidant enzyme determination

Frozen plant leaf (0.1 g) was well grinded in chilled pestle mortar at low temperature (4 °C) in 2mL of 50mM phosphate buffer having 7.8 pH. This homogenized concoction was then centrifugated at 13,000 rpm for 10 min. The resultant supernatant was then preserved at -20 °C in Eppendorf and used to determine various biochemical parameters like antioxidant enzyme activities on a protein basis.

For catalase and peroxidase activity determination [42] and [43], protocols were followed, respectively. Superoxide-dismutase enzymatic activity was calculated according to [44] protocol. Soluble proteins and total amino acids were quantified by [45] and [46] protocols.

Determination of MDA and H2O2

Malondialdehyde (MDA) was estimated to be following [47] protocol. A homogenized mixture was prepared by combining 10mL TCA (0.1% w/v) and 0.5 g leaf sample and then centrifuged for ten minutes at 12,000 rpm. Thiobarbituric acid (0.5%) was prepared in 20% TCA. The supernatant (1mL) was mixed with 4mL of thiobarbituric acid and placed at 95 °C in a water bath for half an hour. Wavelengths 532 and 600 nm were used to determine the optical density of the mixture.

Evaluation of hydrogen peroxide (H2O2) contents was done by using the protocol of [48]. Trichloroacetic acid (5mL; 0.1% w/v) was used to grind 0.5 g fresh leaf sample; the mixture was then centrifuged for 15 min at 12,000 rpm. The supernatant was separated and mixed with 0.5mL phosphate buffer (pH 7.8), and 1mL KI, vortexed, and 390 nm wavelength were set to check the absorbance of the mixture.

Non-enzymatic antioxidants determination.

Ascorbic acid

For ascorbic acid determination [49] protocol was followed. A homogenized mixture was prepared by combining 10mL TCA (0.1% w/v) and 0.5 g leaf sample at low temperature and then centrifuged for ten minutes at 12,000 rpm. In 1mL supernatant, 0.5mL 2% dinitrophenyl hydrazine was added. After adding one drop of 10% thiourea produced in 70% ethanol, the mixture was heated in a water bath for 15 min. Ice was cooled once removed from the water bath, and 1.5mL of 80% H2SO4 was introduced at 0 °C. Transmission density was noted at 530 nm wavelength.

Total soluble phenolic

Fresh leaf (0.2 g) and 2mL 80% acetone were grounded in pestle and mortar using the protocol of [50]. The blend was then centrifugated for 15 min at 12,000 rpm. The supernatant (0.1mL) was put in test tubes beside 1mL distilled water and 0.5mL phenol (Folin Ciocalteu’s) reagent. The mixture was shaken vigorously, and 2.5mL of 20% Na2CO3 was added immediately. Further, 1mL distilled H2O was added, and the mixture was vortexed for 5–10 s and kept for 20 min. After that, the optical density was taken at 750 nm.

Osmolytes determination.

Proline

Fresh leaf material (0.5 g) was ground in 10mL sulphosalicylic acid (3% w/v) and filtered according to the protocol of [51]. In 2mL filtrate, 1mL acid ninhydrin and 1mL glacial acetic acid were introduced and warmed for one hour in a water bath at 100 °C. After that, the test tubes were chilled in an ice bath. Further, 4mL toluene was introduced in tubes and vortexed. The upper layer’s optical density was measured at 520 nm.

Total soluble sugars

Protocol of [52] was followed to approximate total soluble sugar contents. To do this, a pestle and mortar ground 0.1 g of fresh leaf material in 10 mL of distilled water. Then, this ethanolic aliquot (0.1 ml) was thoroughly vortexed with 3mL Anthrone reagent (freshly prepared). The temperature was set to 95 °C in a water bath, and the mixture was for 15 min. Then, after cooling, the absorbance was at 625 nm.

Glycine betaine

[53] was followed for the calculation of glycine betaine contents. Fresh leaf (0.2 g) was homogenized in distilled water (4 mL) and centrifugated at 12,000 rpm for 10 min. The supernatant (1mL) was mixed with 1mL 2NH2SO4, and extract (0.5mL) from it was added to a test tube. 0.2mL KI3 was introduced to it and kept in ice. After ninety minutes, 1.4mL distilled water and 6mL chilled 1-2-dichloromethane was added. At that time, the visualization of the two layers was very distinct. The absorbance of the lower layer was noted at 365 nm.

Statistical analysis

Statistics 8.1 software was applied to scrutinize the final data outcome using the factorial ANOVA test. The variance evaluation was done to estimate the effects of drought, cultivars, melatonin treatment, and their interactions on various parameters. P-values were used to describe the significance of each factor, i.e., P < 0.001, P < 0.01, and P < 0.05 with comparable significance levels ***, **, and *. For means comparisons analysis, the least significant difference (LSD) (P < 0.05) was applied.

Results

Germination percentage

Drought stress (DS) drastically declined seed germination in both wheat cultivars, i.e., Akber-19 and Fakhar-e-Bhakkar, as compared to the control (NoDS) (Figure:1). In terms of germination percentage, the reaction of both cultivars differs notably. Drought stress (DS) caused a 46.15% decline in the gemination percentage of cv. Akber-19 and − 21.48% decline in cv. Fakhar-e-Bhakkar as contrasted to control. Conversely, seed priming with various melatonin levels significantly enhanced the germination percentage in both wheat cultivars under control and drought environments. Under control (NoDS), a maximum increase in germination percentage of both cultivars was observed in seeds primed with M1 treatment. In cv. Akber-19 M1 treatment enhanced the germination percentage by 18.75%, while in Fakhar-e-Bhakkar 22.22% germination rate was noted. Whereas under drought stress, M2 treatment caused a substantial boost in the germination percentage of both cultivars, 54.84% and 33.33% more germination rate was observed in Akber-19 and Fakhar-e-Bhakkar, respectively (Fig. 1A).

Leaf relative water content (%)

Both wheat cultivars ' leaf relative water contents (RWC) showed considerable reduction under drought (DS) stress. Cultivar Akber-19 experienced more decline (-33.89%) than cv. Fakhar-e-Bhakkar (-12.02%) under stress environment. Under no drought stress, seeds pre-soaking with different concentrations of melatonin enhanced RWC percentage considerably; treatments M1 and M2 had a maximum increasing effect on RWC of leaf in both cultivars, 10.3%, 9.7% in Akber-19 and 7.55%, 7.26% in Fakhar-e-Bhakkar respectively. However, under drought stress (DS), considerable progress in leaf water content percentage was recorded in seeds pre-soaked with different levels of melatonin, and among them, M2 treatment represented maximum RWC (%), i.e., 22.38%, 13.28% in Akber-19 and Fakhar-e-Bhakkar respectively, as compared to seeds primed with normal distilled water under stress circumstances (Fig. 1B).

Fig. 1
figure 1

The effect of seed priming with different levels of melatonin (M0-M5) on germination percentage (A) and relative water content percentage (B) on two wheat cultivars under NoDS (control) and 35% DS (drought stress). Bars denote mean ± SE (n = 4); evaluated using LSD at p ≤ 0.05 significance. Letters on error bars denote substantial differentiation among treatments

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Shoot fresh weight

Drought stress (DS) had a noteworthy declining outcome on shoot fresh weight of both wheat cultivars, -54.49% in Akber-19 and − 44.69% in Fakhar-e-Bhakkar, but seed pre-sowing treatment with different levels of melatonin considerably boosted the shoot fresh mass under drought stress (DS) and control (NoDS) than hydro-primed seeds (M0). Under no drought stress (NoDS) conditions, seed soaking with M1 = 1mgL− 1melatonin treatment considerably enhanced shoot fresh weight of both cultivars, 20.89% and 23.48% in Akber-19 and Fakhar-e-Bhakkar respectively. However, under DS conditions, M2 = 2mgL− 1 treatment of melatonin triggered a considerable increase in shoot fresh weight of Akber-19 (52.70%) and Fakhar-e-Bhakkar (39.12%) (Fig. 2A).

Shoot dry weight

The imposition of drought stress (DS) drastically diminished the shoot dry weight of both sensitive and tolerant wheat cultivars. At the same time, all melatonin pre-sowing seed treatments effectively increased the shoot dry mass of both cultivars under stress (DS) and no stress (NoDS). However, seed pre-soaking with M1 = 1mgL− 1 was more effective in increasing shoot dry weight under control conditions as cultivar Akber-19 showed 34.14% while Fakhar-e-Bhakkar displayed a 30.14% increase under control. While under DS, a maximum increase in shoot dry weight was observed in M2 = 2mgL− 1 treatment of melatonin (Fig. 2B).

Root fresh weight

Pre-sowing seed soaking treatment with different melatonin levels significantly enhanced the root fresh weight of both wheat cultivars (Akber-19 and Fakhar-e-Bhakkar) under control (NoDS) as well as drought (DS). Melatonin level M1 = 1mgL− 1 was more effective in enhancing root fresh weight under control than all other melatonin treatments, as a 12.73% increase was observed in Akber-19 and a 15% increase in Fakhar-e-Bhakkar. A remarkable variation among cultivars displayed that cv. Akber-19 represented higher root fresh weight under control as compared to cv. Fakhar-e-Bhakkar. Whereas, under DS conditions, melatonin treatment M2 = 2mgL− 1 displayed a significant increase in both cultivars, 20.73% (Akber-19) and 36.36% (Fakhar-e-Bhakkar) in root fresh weight as compared to their respective controls (Figure:2-C).

Root dry weight

Drought stress (DS) significantly declined root dry weight in both wheat cultivars, i.e., Akber-19 and Fakhar-e-Bhakkar, as compared to the control (NoDS) (Fig. 2-D). Drought stress (DS) caused a -25.0% decline in root dry weight of cv. Akber-19 and − 30.77% decline in cv. Fakhar-e-Bhakkar in comparison to control. Conversely, seed priming with various melatonin levels significantly enhanced the root dry mass in wheat cultivars under control and drought circumstances (Fig. 2-D). Under control (NoDS), the largest rise in root dry weight of both cultivars was monitored in seeds primed with M1 = 1mgL− 1 treatment. In cv. Akber-19 M1 treatment enhanced the root dry weight by 34.78%, while in Fakhar-e-Bhakkar, 32% more was noted. Whereas, under drought stress, M2 = 2mgL− 1 treatment produced a substantial increase in root dry weight of both cultivars, 29.41% and 23.53% more root dry weight was observed in Akber-19 and Fakhar-e-Bhakkar, respectively.

Shoot length

The shoot length of both wheat cultivars experienced considerable reduction (P ≤ 0.001) under drought stress (DS); a 23.67% decline was observed in cultivar Akber-19 and − 27.13% in cv—Fakhar-e-Bhakkar as compared to control. Under no control (NoDS) seed priming treatment, M1 = 1mgL− 1 and M2 = 2mgL− 1 were better (i.e., 13.5% and 13%, respectively) in increasing shoot length in Akber-19 (Fig.2-E). However, in Fakhar-e-Bhakkar, the M1 treatment displayed the maximum increase. Conversely, under drought stress (DS), a substantial rise in shoot length was noted in seeds primed with different levels of melatonin, and among them, M2 treatment represented the maximum increase, i.e., 17.92% in both cultivars as compared to seeds primed with normal distilled water under stress circumstances.

Root length

Imposition of drought stress (DS) significantly reduced the root length of both cultivars, i.e., Akber-19 (-26.47%) and Fakhar-e-Bhakkar (-19.05%) than the control ones. At the same time, all melatonin pre-sowing seed treatments effectively increased the root length of both cultivars under stress (DS) and under no stress (NoDS). However, seed pre-soaking with M1 = 1mgL− 1 was more effective in increasing root length under control conditions (Figure:2-F) as cultivar Akber-19 showed 17.78%. Fakhar-e-Bhakkar displayed a 13.26% increase under control compared to other melatonin treatments. While under DS, a maximum increase in root length was observed in M2 = 2mgL− 1 treatment of melatonin, where 20.93% and 18.49% more growth was observed in Akber-19 and Fakhar-e-Bhakkar respectively, as compared to distilled water primed seeds.

Fig. 2
figure 2

The impact of seed priming with various levels of melatonin (M0-M5) on shoot fresh weight (A), shoot dry weight (B), root fresh weight (C), root dry weight (D), shoot length (E), root length (F) on two wheat cultivars under NoDS (control) and 35% DS (drought stress). Bars denote mean ± SE (n = 4); evaluated using LSD at p ≤ 0.05 significance. Letters on error bars denote substantial differentiation among treatments

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Leaf area

Drought stress (DS) implementation had an inhibitory effect on the overall growth of both wheat cultivars. Similarly, the leaf area declined significantly under stress; however, this decline was more prominent in Fakhar-e-Bhakkar as it showed a -48.98% decline under stress compared to its respective control (M0). The highest value of leaf area under control (NoDS) was displayed by cv. Fakhar-e-Bhakkar and seed priming with melatonin treatments M1 and M2 further caused an upsurge in leaf area by 14.58% and 14.33%, respectively. Whereas in cv. Akber-19, M1 melatonin treatment increased (16.40%) leaf area more effectively. Under DS, a considerable boost in leaf area was monitored in melatonin M1, M2, and M3 treatments in both cultivars. However, the M2 effect was 25.18% in Akber-19, and a 29.21% increase in Fakhar-e-Bhakkar was noted under drought stress (DS) (Fig. 3A).

Chlorophyll a

Chlorophyll-a content of both wheat cultivars experienced considerable reduction (P ≤ 0.001) under drought stress (DS), -an 11.95% decline was observed in cultivar Akber-19 and − 11.74% in cv—Fakhar-e-Bhakkar as compared to control. M2 treatment enhanced chlorophyll-a (17.91%) in Akber-19 under stress more effectively. However, under no drought stress conditions (NoDS), seed priming treatment M1 = 1mgL− 1 was better in increasing (18.34%) chlorophyll-a content in Akber-19. Meanwhile, under NODS in Fakhar-e-Bhakkar, the M1 treatment displayed a maximum increase (15.63%). Conversely, under drought stress (DS), a considerable rise in chlorophyll-a was noted in seeds primed with different levels of melatonin, and among them, M2 treatment represented the maximum increase, i.e., 17.03% in Fakhar-e-Bhakkar as compared to seeds primed with normal distilled water under stress environment (Fig. 3B).

Chlorophyll b

The imposed drought stress (DS) significantly reduced chlorophyll b contents of both sensitive (-29.09%) and tolerant (-27.85%) wheat cultivars compared to the control ones. All melatonin pre-sowing seed treatments efficiently improved chlorophyll b contents of both cultivars under stress (DS) and no stress (NoDS). However, seed pre-soaking with M1 = 1mgL− 1 was more efficient in improving chlorophyll b content under a controlled environment, as cultivar Akber-19 showed a 13.84% increase. In comparison, Fakhar-e-Bhakkar displayed a 20.07% increase respectively under control. While under DS, the highest rise in chlorophyll b was observed in M2 = 2mgL− 1 treatment of melatonin, where 24.30% more chlorophyll b was observed in Akber-19 and 23.11% in Fakhar-e-Bhakkar as compared to distilled water primed seeds. Meanwhile, in Fakhar-e-Bhakkar, M1 and M2 treatment displayed a similar rise (19.83%) under stress (Fig. 3C).

Chlorophyll a/b

Pre-sowing seed treatment with different melatonin levels significantly decreased the chlorophyll a/b ratio of both wheat cultivars (Akber-19 and Fakhar-e-Bhakkar) under control (NoDS) as well as under drought (DS). However, in Akber-19, melatonin levels M1 = 1mgL− 1 and M2 = 2mgL− 1 displayed remarkable variation, showing a 1.6% and 1.7% increase in chlorophyll a/b ratio under control. In Fakhar-e-Bhakkar, all treatments displayed a decreasing effect under control (NoDS). Whereas, under DS conditions, melatonin treatment M2 = 2mgL− 1 in Akber-19 and M3 = 3mgL− 1 in Fakhar-e-Bhakkar displayed a significant decrease, -9.09% and − 15.95%, respectively, in chlorophyll a/b ratio (Fig. 3D).

Total chlorophyll

Drought stress (DS) significantly declined total chlorophyll in wheat cultivars i.e., Akber-19 and Fakhar-e-Bhakkar, compared to control (NoDS). Drought stress (DS) caused a 20.39% decline in total chlorophyll of cv. Akber-19 and − 18.39% decline in cv. Fakhar-e-Bhakkar as compared to control. However, seed priming with different melatonin levels extensively enhanced the total chlorophyll contents in both wheat cultivars under control and drought conditions. Under control (NoDS), a substantial rise in total chlorophyll of both cultivars was monitored in seeds primed with M1 = 1mgL− 1 treatment. In cv. Akber-19, M1 treatment enhanced the total chlorophyll by 16.28%, while in Fakhar-e-Bhakkar, 17.67% more chlorophyll was noted under the same treatment. Whereas, under drought stress, M2 = 2mgL− 1 treatment produced a substantial increase in total chlorophyll of both cultivars, 20.85% and 19.65% more total chlorophyll was observed in Akber-19 and Fakhar-e-Bhakkar respectively (Fig. 3E).

Carotenoid contents

Drought stress (DS) implementation had an inhibitory effect on the overall growth of both wheat cultivars. Similarly, carotenoid contents declined significantly under stress. However, this decline is more prominent in Fakhar-e-Bhakkar as it showed a -19.07% decline in carotenoids under stress compared to its respective control (M0). The maximum value in carotenoids under control (NoDS) was displayed by cv. Akber-19 and seed priming with melatonin treatment, specifically M1, further caused an upsurge in leaf carotenoids by 11.08%, similarly in CV. Fakhar-e-Bhakkar M1 treatment displayed the highest (14.38%) increasing effect in leaf carotenoids. Under DS, a substantial increase in leaf carotenoids of both cultivars was monitored in all melatonin treatments. However, the M2 effect was above all, 16.11% in Akber-19, and 21.12% increase in Fakhar-e-Bhakkar was noted under drought stress (DS) (Fig. 3F).

Fig. 3
figure 3

The effect of seed priming with various levels of melatonin (M0-M5) on leaf area (A), chlorophyll a (B), chlorophyll b (C), chlorophyll a/b (D), total chlorophyll (E), carotenoids (F) on two wheat cultivars under NoDS (control) and 35% DS (drought stress). Bars denote mean ± SE (n = 4); evaluated using LSD at p ≤ 0.05 significance. Letters on error bars denote substantial differentiation among treatments

Full size image

Proline

Drought stress (DS) significantly enhanced proline contents in both wheat cultivars, i.e., Akber-19 (12.71%) and Fakhar-e-Bhakkar (13.47%), as compared to control (NoDS) (Figure:4-A4-A). Similarly, seed priming with various melatonin levels considerably enhanced the proline contents in cv wheat cultivars under control and drought environments. Akber-19, all melatonin treatments almost equally enhanced proline contents under control (NoDS); however, the maximum increase (8.83%) was displayed by the M1 treatment, likewise in cv. Fakhar-e-Bhakkar maximum increase (10.17%) in proline contents was observed in seeds primed with M1 treatment under NoDS. Though, under DS conditions, cv. Akber-19 experienced increased proline contents under all melatonin treatments, while M2 treatment displayed maximum (16.54%) enhancement, relatedly in cv. Fakhar-e-Bhakkar, M2 treatment displayed a maximum (11.37%) proline value under stress (DS) compared to all other seed priming treatments (Fig. 4A).

Soluble sugars

Pre-sowing seed soaking treatment with different melatonin levels significantly enhanced soluble sugar contents (SS) of both wheat cultivars under control (NoDS) as well as under drought (DS). Melatonin treatment M1 was more effective in enhancing (8.46%) soluble sugar contents under control than other melatonin treatments in Akber-19. Similarly, in the Fakhar-e-Bhakkar melatonin treatment, M1 = 1mgL− 1 displayed a maximum increase (10.56%) in soluble sugar contents under NoDS. A remarkable variation among cultivars was also observed. However, under DS conditions, melatonin treatment M2 = 2mgL− 1 displayed a significant increase in soluble sugars, 13.98% in Akber-19 and 8.06% in Fakhar-e-Bhakkar (Fig. 4B).

Total soluble proteins

The imposition of drought stress (DS) and changes in total soluble proteins (TSP) is cultivar-specific. As in our findings, Akber-19 showed a very slight increase (2.05%) in soluble proteins under DS conditions than control (NoDS). Whereas in cv. Fakhar-e-Bhakkar, there was a 16.95% increase in total soluble proteins under DS compared to the control group. However, under no drought stress conditions (NoDS), seed pre-soaking treatment with different melatonin concentrations showed a considerable increase in soluble proteins in both cultivars.

Similarly, under drought stress (DS) environments, a noteworthy increase was observed in total soluble proteins in seeds pre-soaked with different levels of melatonin, and among them, M2 treatment represented the maximum increase, i.e., 21.38% in Akber-19 and 16.49% in cv. Fakhar-e-Bhakkar compared to seeds pre-soaked with normal distilled water under stress (DS) circumstances. However, three levels of melatonin treatment, M1, M2, and M3, were more effective in enhancing total soluble proteins under stress conditions (Fig. 4C).

Total free amino acids

Drought stress (DS) implementation increased the total free amino acids (TFAA) of both wheat cultivars in seeds pre-soaked with distilled water, 9.26% in Akber-19 and 35.22% increase in cv. Fakhar-e-Bhakkar was observed. In melatonin-primed seeds, total free amino acid contents enhanced significantly under stress. However, this increase is more prominent in M2 = 2mgL− 1 treatment, as a 17.2% increase was observed in Akber-19 and 15% in cv. Fakhar-e-Bhakkar is under stress as compared to its respective control (M0). The highest value in total free amino acids in cv. Akber-19 under control (NoDS) was displayed by M1 melatonin treatment (i.e., 19.43%), similarly in CV. Fakhar-e-Bhakkar M1 treatment was more effective in increasing (27.01%) total free amino acids under control (NoDS) conditions (Fig. 4D).

Glycinebetaine

Drought stress (DS) significantly enhanced glycinebetaine (GB) contents in both wheat cultivars, i.e., Akber-19 (12.86%) and Fakhar-e-Bhakkar (24.72%), as compared to control (NoDS). Conversely, seed priming with certain melatonin levels significantly enhanced glycinebetaine contents in both wheat cultivars under control and drought circumstances. In cv. Akber-19 melatonin treatment M1 enhanced glycinebetaine contents 10.44% under control (NoDS), similarly in cv. Fakhar-e-Bhakkar maximum increase (9.42%) in glycinebetaine contents was observed in seeds primed with M1 treatment under NoDS. However, under DS conditions, cv. Akber-19 experienced an increase (11.75%) in glycinebetaine content under M2 melatonin treatment, relatedly in cv. Fakhar-e-Bhakkar, M2 treatment displayed 8.15% more glycinebetaine contents under stress (DS) than all other melatonin treatments (Fig. 4E).

Soluble phenolics

Soluble phenolics of both wheat cultivars experienced a considerable increase (P ≤ 0.001) under drought stress (DS); an 11.91% increase was observed in cultivar Akber-19 and 6.75% in cv. Fakhar-e-Bhakkar as compared to control. M1 and M2 treatments enhanced soluble phenolics (16.39% and 18.46%) in Akber-19 under DS. However, under no drought stress conditions (NoDS), seed priming treatments M1 = 1mgL− 1 were better in increasing (12.56%) soluble phenolics in Akber-19 (Fig.4-F). Similarly, under NODS conditions, the Fakhar-e-Bhakkar M1 treatment displayed a maximum increase (15.48%). Conversely, under drought stress (DS), a substantial rise in soluble phenolics was noted in seeds primed with different levels of melatonin, and among them, M2 treatment represented the maximum increase, i.e., 11.48% in Fakhar-e-Bhakkar as compared to seeds primed with normal distilled water under stress circumstances (Fig. 4F).

Fig. 4
figure 4

The effect of seed priming with various levels of melatonin (M0-M5) on proline (A), soluble sugars (B), total soluble proteins (C), total free amino acids (D), glycine betaine (E), soluble phenolics (F) on two wheat cultivars under NoDS (control) and 35% DS (drought stress). Bars denote mean ± SE (n = 4); evaluated using LSD at p ≤ 0.05 significance. Letters on error bars denote substantial differentiation among treatments

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Malondialdehyde

Drought stress (DS) significantly enhanced malondialdehyde contents in wheat cultivars i.e., Akber-19 and Fakhar-e-Bhakkar, compared to the control (NoDS). Drought stress (DS) caused a 34.34% increase in malondialdehyde of cv. Akber-19 and 14.44% increase in cv. Fakhar-e-Bhakkar as compared to control. However, seed priming with various melatonin levels considerably decreased the malondialdehyde (MDA) contents in wheat cultivars under control and drought environments. Under control (NoDS), a considerable reduction in malondialdehyde contents of both cultivars was noted in seeds primed with M1 = 1mgL− 1 treatment. In cv. Akber-19, -13.05% decrease in the malondialdehyde contents, while in Fakhar-e-Bhakkar, -26.00% decline was noted under the same treatment. Whereas, under drought stress, M2 = 2mgL− 1 treatment caused a significant decline in malondialdehyde contents of both cultivars than their respective controls, -41.29% and − 22.03% decline was observed in Akber-19 and Fakhar-e-Bhakkar respectively (Fig. 5A).

Hydrogen peroxide

Drought stress (DS) significantly enhanced hydrogen peroxide contents in both wheat cultivars, i.e., Akber-19 (29.77%) and Fakhar-e-Bhakkar (14.84%), as compared to control (NoDS). Seed priming with various melatonin levels considerably declined the H2O2 contents in wheat cultivars under control and drought (DS) environments. In cv. Akber-19 and cv. Fakhar-e-Bhakkar melatonin treatment M1 = 1mgL− 1 significantly decreased H2O2 contents by 16.60% and 19.91% under control (NoDS). However, under DS conditions, cv. Akber-19 experienced a decrease in H2O2 contents under all melatonin treatments, while M2 treatment displayed a maximum (--31.17%) decline similar to in cv. Fakhar-e-Bhakkar, M2 treatment displayed − 18.07% less H2O2 under stress (DS) than all other seed priming treatments (Fig. 5B).

Ascorbic acid

Ascorbic acid contents of both wheat cultivars experienced a considerable increase (P ≤ 0.001) under drought stress (DS); a 13.44% increase was observed in cultivar Akber-19 and 18.85% in cv. Fakhar-e-Bhakkar as compared to control. Apparently, under NoDS conditions, M1 treatment enhanced ascorbic acid contents in Akber-19 (13.75%) and Fakhar-e-Bhakkar (23.49%). Meanwhile, M2 treatment displayed a maximum increase of 8.4% under DS in cv. Fakhar-e-Bhakkar while in cv. Akber-19’s M1 and M2 melatonin treatments significantly enhanced 13.34% and 15.56% ascorbic acid contents, respectively (Fig. 5C).

Peroxidase

Melatonin pre-sowing seed treatment significantly enhanced peroxidase activity in wheat cultivars under NoDS and drought-stress circumstances. Both cultivars displayed a rise in POD activity under drought stress, i.e., a 5.78% increase was seen in Akber-19, whereas in Fakhar-e-Bhakkar, the increase was 30.86% compared to control (NoDS). Moreover, all melatonin treatments under DS caused an elevation in POD activity. In cv. Akber-19, M2 treatment displayed a significant elevation of 48.36%, likewise in cv. Fakhar-e-Bhakkar M2 treatment caused a maximum elevation of 39.02% more than all other treatments under drought stress than their respective controls. Under NoDS conditions in cv. Akber-19 melatonin treatments M1, M2, M3, M4 and M5 displayed following elevations 24.19% > 18.5% > 11.89% > 10.24% and 7.68% maximum to minimum respectively. Similarly, in cv. Fakhar-e-Bhakkar, the elevation in POD activity from maximum to minimum is 35.29% > 28.82% > 21.33% > 19.33% and 8.33% compared to their respective control. Maximum rise in peroxidase activity was detected in seeds primed with M1 treatment under NoDS (Fig. 5D).

Catalase

Drought stress (DS) significantly enhanced catalase activity in both wheat cultivars; however, cv. Fakhar-e-Bhakkar displayed a rise in catalase (CAT) activity, i.e., 45.81 than Akber-19 (9.97%) as compared to respective controls (NoDS). Moreover, under DS all melatonin treatments caused further elevation in CAT activity. In cv. Fakhar-e-Bhakkar, under drought stress, M2 treatment displayed a significant elevation of 34.7%, similarly in cv. Akber-19, M2 treatment caused a maximum increase of 39.78% in CAT activity compared to all other treatments under NoDS conditions in cv. Akber-19 melatonin treatment M1 displayed 32.04% elevation, similarly in cv. Fakhar-e-Bhakkar, the elevation in CAT activity was more significant in M1 and M2 treatments, i.e., 49.06% and 44.45%, respectively, under NoDS. However, the maximum rise in catalase activity was monitored in seeds primed with M1 treatment under NoDS (Fig. 5E).

Superoxide dismutase

Drought stress (DS) implementation had an increasing effect on superoxide dismutase (SOD) activity of both wheat cultivars in seeds pre-soaked with distilled water, 24.63% in Akber-19 and 31.63% increase in cv. Fakhar-e-Bhakkar was observed. Whereas in melatonin-primed seeds, SOD activity was enhanced significantly under stress (DS); hence this increase was more prominent in cv. Akber-19, where the rise in M2 treated seeds was 44.37% more than its respective control (M0). Similarly, in cv. Under stress, the fakhar-e-Bhakkar maximum increase (16.36%) was observed in M2-treated seeds. The highest value of SOD activity in cv. Akber-19 under control (NoDS) was displayed by M1 melatonin treatment at 25.55%, similarly in cv. Fakhar-e-Bhakkar M1 treatment was more effective in increasing (22.89%) SOD activity under control (NoDS) conditions. These results indicated that both varieties were affected by drought stress, as evidenced by the increase in SOD activity. Cv. Fakhar-e-Bhakkar displayed the most pronounced increase, signifying its ability to tolerate drought stress.

Conversely, Akber-19 showed less increase in SOD activity, suggesting lesser resilience to drought stress. However, melatonin treatment had a pronounced effect on the antioxidant enzyme activities of Akber-19 and Fakhar-e-Bhakkar, which aids in increasing tolerance against stress. These outcomes emphasize the importance of selecting appropriate melatonin concentrations for seed pre-treatment before cultivation under DS conditions. Melatonin treatments such as M1 = 1mgL− 1 and M2 = 2mgL− 1 proved to be more efficient in easing the damaging impacts of DS on wheat productivity. Whereas higher concentrations of melatonin have a less profound impact on wheat cultivars under study in stress (DS) conditions, advising that we should optimize melatonin concentrations for wheat crop growth (Fig. 5F).

Fig. 5
figure 5

The impact of seed priming with various levels of melatonin (M0-M5) on malondialdehyde (MDA) (A), hydrogen peroxide (H2O2) (B), ascorbic acid (C), peroxidase (D), catalase (E), superoxide dismutase (F) on two wheat cultivars under NoDS (control) and 35% DS (drought stress). Bars denote mean ± SE (n = 4); evaluated using LSD at p ≤ 0.05 significance. Letters on error bars denote substantial differentiation among treatments

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Discussion

Morpho-physiological responses

Drought stress (DS) significantly alters plant growth and development, with biochemical and physiological changes depending on the drought sensitivity or tolerance of the cultivar [54]. The present study aimed to evaluate exogenous melatonin’s role in improving wheat’s morphological and physiological parameters under DS. As widely documented [55,56,57], melatonin is defensive in regulating plant growth under abiotic stresses. Under DS, wheat plants naturally increase antioxidant activity to evade oxidative stress, yet exogenous melatonin treatment further enhances this tolerance, even in sensitive cultivars [58].

The results demonstrated that DS led to substantial inhibitions in shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW), root dry weight (RDW), shoot length (SL), root length (RL), and leaf area (LA), likely due to restricted cell progression and division caused by limited water availability. However, melatonin seed priming mitigated these inhibitory effects, improving germination percentage, relative water content (RWC), biomass, and leaf area, critical parameters for assessing drought tolerance. The enhancement in seed germination and plant growth can be attributed to melatonin’s ability to alter embryonic physiology, accelerating the action of hydrolytic enzymes thus promoting rapid germination. These findings are consistent with previous research by [59].

Biochemical responses and photosynthetic pigments

Melatonin’s role in maintaining photosynthetic efficiency under DS was evident in our study. Drought stress reduced chlorophyll a, b, and total chlorophyll (Chl.), which could be associated with thylakoid membrane impairment and degradation of photosynthetic pigments. However, melatonin seed priming lessened these adverse effects by enhancing the photosynthesis of photosynthetic pigments, safeguarding photosystem II, and maintaining chloroplast structure, cell expansion, and integrity of the cell wall. Melatonin has been shown to improve leaf chlorophyll content, gas exchange, and photo-chemical efficiency under stress [60], findings that align with our results.

The protective role of melatonin in maintaining photosynthesis under DS is further supported by its ability to modulate stomatal behavior and enhance water retention, as demonstrated by increased leaf area and chlorophyll contents. Previous studies have also demonstrated melatonin’s ability to protect photosynthetic pigments, maintaining photosynthesis under abiotic stresses [61, 62].

Non-enzymatic antioxidants and osmolytes

Drought stress triggers the formation of reactive oxygen species (ROS), leading to oxidative stress and subsequent damage. In response, plants produce non-enzymatic antioxidants such as phenolics, carotenoids, proline, and ascorbic acid, which scavenge ROS and mitigate damage [63]. In our study, exogenous melatonin treatment enhanced the accumulation of these non-enzymatic antioxidants, particularly proline, soluble sugars, and glycine betaine, helping to improve the osmotic potential of the cells. These findings align with earlier research, highlighting melatonin’s role in enhancing proline and soluble sugar accumulation under DS, as seen in various species [64, 65].

The accumulation of proline, in particular, is crucial for stabilizing membrane structures and protecting plants from oxidative damage. Additionally, soluble proteins, which play a key role in osmoregulation, increased with melatonin treatment. These results further suggest that melatonin enhances the plant’s osmotic balance, promoting its survival under drought conditions. The role of melatonin in osmotic regulation is well-documented, with similar results observed in various species under abiotic stress [66].

Enzymatic antioxidant activities

Under DS, plants increase the activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to counteract the accumulation of ROS and mitigate oxidative damage [67]. In the present study, melatonin seed priming significantly enhanced the activity of these antioxidant enzymes. SOD acts as the first line of defense by catalyzing the conversion of superoxide radicals (O2-) into less harmful molecules like H2O2, which are subsequently broken down by CAT and POD into water molecules, thereby reducing oxidative stress.

Melatonin, known for its universal antioxidant properties, acts directly as a ROS scavenger and triggers other antioxidant defense systems [66]. The observed decrease in H2O2 and malondialdehyde (MDA) levels in melatonin-treated wheat plants further supports its role in alleviating oxidative stress. MDA, a marker of lipid peroxidation, can lead to membrane decomposition and cellular damage. In our study, melatonin reduced MDA accumulation, preserving cellular integrity under DS. These findings are consistent with studies conducted on cotton [64], C. cathayensis [68], and M. oleifera, further corroborating melatonin’s role in enhancing stress tolerance [32, 54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81].

Conclusions and recommendations

It is concluded that 2 mg L− 1 melatonin seed priming effectively improves wheat cv. Akber-19 and Fakhar-e-Bhakkar growth, chlorophyll contents under drought stress. Better regulation of osmolytes and antioxidants in wheat cv. Akber-19 and Fakhar-e-Bhakkar also validated the effectiveness of 2 mg L− 1 melatonin concentration as a seed priming agent for mitigating drought stress. However, it is important to note some limitations. The study focused primarily on the positive effects of 2 mg L⁻¹ melatonin. In contrast, higher concentrations (i.e., 3 mg L⁻¹, 4 mg L⁻¹, and 5 mg L⁻¹) did not produce significant results, and these concentrations require further investigation to determine their broader applicability. For practical applications, using melatonin-primed seeds could be a promising strategy for wheat growers to enhance drought resilience. Future research should focus on larger field trials, optimizing melatonin concentrations across diverse wheat cultivars.

Data availability

All data generated or analyzed during this study are included in this published article.

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Acknowledgements

The data presented in this manuscript is a part of PhD research work of Sehar Shaheen (Scholar) at the Department of Botany, Government College Women University Faisalabad. The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R98), King Saud University, Riyadh, Saudi Arabia, for financial support.

Funding

The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R98), King Saud University, Riyadh, Saudi Arabia, for financial support.

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Authors and Affiliations

  1. Department of Botany, Government College Women University, Faisalabad, Pakistan

    Sehar Shaheen & Irfana Lalarukh

  2. Wheat Research Institute, Ayub Agriculture Research Institute, Faisalabad, Pakistan

    Javed Ahmad

  3. Office Soil and Water Testing Laboratory for Research, Bahawalpur, Punjab, Pakistan

    Syed Ali Zulqadar

  4. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

    Sulaiman Ali Alharbi & Abdullah A. Alarfaj

  5. Department of Environmental Sciences, Woman University Multan, Multan, Punjab, Pakistan

    Misbah Hareem

  6. Department of Botany, Hindu College Moradabad (Mahatma Jyotiba Phule Rohilkhand University Bareilly)-Uttar Pradesh, Moradabad, 24400, India

    Mohammad Javed Ansari

Authors
  1. Sehar Shaheen
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  2. Irfana Lalarukh
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  3. Javed Ahmad
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  4. Syed Ali Zulqadar
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  5. Sulaiman Ali Alharbi
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  7. Abdullah A. Alarfaj
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  8. Mohammad Javed Ansari
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Contributions

Conceptualization, methodology, formal analysis, data curation, and writing-original draft preparation were done by S.S.; I.L.; J.A.; supervision, investigation, validation, and project administration were done by S.S.; I.L.; J.A.; software, writing-review, and editing, A.A.A.; M.H., S.A.A.; S.A.Z.; and M.J.A. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Sehar Shaheen, Irfana Lalarukh or Misbah Hareem.

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We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable. Study protocol must comply with relevant institutional, national, and international guidelines and legislation. Our experiment follows the relevant institutional, national, and international guidelines and legislation.

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Not Applicable.

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Shaheen, S., Lalarukh, I., Ahmad, J. et al. Physio-biochemical mechanism of melatonin seed priming in stimulating growth and drought tolerance in bread wheat. BMC Plant Biol 24, 918 (2024). https://doi.org/10.1186/s12870-024-05639-8

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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