Metabolomic responses of wheat grains to olive mill wastewater and drought stress treatments

Abstract

The present research aimed to assess the metabolomic responses of wheat to olive mill wastewater (OMWW) and drought stress treatments. Wheat plants were cultivated under controlled conditions with the following treatments: control (75% field capacity, FC), OMWW (75 ml L⁻¹), drought stress (40% FC, applied 30 days after sowing), and a combined treatment of OMWW and drought stress. Drought stress alone reduced grain yield by 67%, while the OMWW-treated plants resulted in a 29% reduction under stress relative to the control. OMWW application improved soil properties, enhancing organic matter and nutrient levels. Wheat grains from OMWW-treated plants exhibited higher sugar content and related enzyme activities, indicating improved metabolism, with significant increases in starch, fructose, and glucose levels alongside stable invertase and sucrose phosphate synthase activities. The study also noted substantial changes in amino acids, fatty acids, and phenolic acids in plants subjected to OMWW and drought stress. These modifications indicate OMWW’s capability to influence vital biochemical pathways and boost antioxidant capacities in wheat. In conclusion, OMWW proves to be an effective soil amendment that mitigates drought stress and contributes to the production of nutrient-rich, resilient wheat, underscoring its potential as a sustainable agricultural practice in water-scarce areas.

Introduction

Wheat (Triticum aestivum L.) is one of the most extensively cultivated and important cereal grains grown worldwide, making it a key component of many people’s diets. Its rich nutritional profile, which includes vital macronutrients, dietary fiber, and beneficial compounds, highlights its crucial role in ensuring food security and supporting agricultural success¹. However, abiotic stresses, particularly water scarcity and soil salinity, pose considerable challenges to wheat cultivation, negatively affecting yield and grain quality. Drought stress triggers various physiological and biochemical responses in plants^1,2. Drought stress significantly affects wheat by altering key physiological and cellular processes crucial for plant survival. When exposed to drought, wheat experiences changes in turgor pressure, cell wall stiffness, membrane stability, and fluid volume, all of which can hinder growth and affect overall crop viability³. Turgor pressure diminishes due to water loss, leading to reduced cell expansion, while membrane stability is compromised due to oxidative stress and increased reactive oxygen species (ROS)⁴. To mitigate these impacts, wheat adapts by enhancing cell wall stiffness and modifying membrane lipid composition, alongside boosting antioxidant defenses to maintain cellular functions¹. The plant’s responses are further linked to genetic and metabolic regulatory processes, prominently involving signaling pathways activated by abscisic acid (ABA), which regulates stomatal closure and promotes osmotic adjustment through specific gene activation^1,5. These intricate interactions between physiological changes and genetic regulation highlight the importance of understanding wheat’s adaptive mechanisms to drought stress. Integrating this knowledge into agricultural practices is essential for developing resilient wheat varieties and improving crop management strategies. Metabolomic studies have emerged as crucial tools in understanding plant stress tolerance mechanisms. Such studies offer insights into how key metabolic pathways are modulated under adverse conditions^6,7. The studies provide detailed profiles of sugars, organic acids, amino acids, and antioxidants, highlighting how plants adapt to stress at a molecular level⁸. The ability to identify specific biochemical changes offers valuable opportunities for developing targeted interventions to enhance stress resilience and crop productivity.

One promising approach to address drought stress in plants is using organic waste, mainly olive mill wastewater (OMWW), which has gained significant interest for its ability to promote plant growth and resilience⁹. OMWW, a byproduct of olive oil production, is abundant in organic matter, essential nutrients, and bioactive compounds, including antioxidant molecules and phenolic acids¹⁰. Moreover, OMWW has been shown to modulate critical metabolic pathways, including those associated with sugar metabolism, antioxidant enzyme activity, and amino acid synthesis, which are pivotal in improving plant stress tolerance and grain quality^10,11. Researchers have proposed using OMWW as an organic biofertilizer to help restore soil fertility and boost agricultural production¹¹. Given its nutrient-rich profile, incorporating OMWW into agricultural management may offer a sustainable solution to enhance plant growth in challenging environments, positively influencing both reproductive and vegetative characteristics^9,10.

On the other hand, water scarcity increasingly poses a significant challenge to sustainable development, as rising urbanization and industrialization are leading to heightened water demand, exacerbating the disparity between supply and demand^12,13. This situation compels the agricultural sector to minimize its reliance on freshwater and explore alternative water sources. Treated wastewater emerges as a viable solution for addressing agricultural water needs, conserving freshwater resources for domestic and industrial applications, promoting enhanced socio-economic conditions, and supporting sustainable development initiatives^13,14.

In the current research, we examined the combined effects of OMWW application and drought stress on the metabolomic profiles of wheat grains. We hypothesized that applying OMWW would mitigate the negative impacts of drought stress, resulting in modifications to the composition of wheat grains. The objective was to evaluate the levels of phytochemicals, including flavonoid and phenolic compounds and antioxidant capacity, across various treatment conditions. Our findings aim to enhance understanding of the interactions between organic amendments and plant stress tolerance mechanisms, thereby informing sustainable agricultural practices designed to improve crop quality and yield. While this research focuses on wheat, the findings have broader implications for other cereal crops and agricultural systems facing similar environmental constraints. By integrating organic amendments like OMWW into agricultural practices, the potential exists to enhance food security and sustainability in regions most vulnerable to climate-induced stresses. By investigating the metabolomic responses of wheat grains subjected to OMWW and drought stress, this research underscores the potential of utilizing organic waste to enhance crop resilience against abiotic stressors.

Results

Plant traits

The effects of drought stress and OMWW treatments on yield and yield component parameters were analyzed, revealing significant differences (Figure. 1). In this regard, the greatest grain yield per plant was observed in the OMWW treatment, recording an average of 2.8 g plant^-1, representing a non-significant shift in comparison with the control plants (p ≥ 0.05). In contrast, the grain yield under drought stress was significantly reduced to 0.9 g plant^-1, resulting in a 66.9% decrease compared to control (p < 0.05). When OMWW was combined with stress, the grain yield was also lower, showing a 28.8% reduction compared to the control (p < 0.05). The control group and OMWW-treated plants showed the longest ear length. Under drought stress, without and with OMWW treatments, ear length significantly decreased by 57.6% and 47.9% compared to the control, respectively (p < 0.05). Control and OMWW-treated plants had a higher average of spikelets per ear. In contrast, drought stress caused a significant drop, equating to a 39.7% reduction (p < 0.05). The combination of OMWW and stress resulted in a 21.6% decrease from the control treatment (p < 0.05). The number of grains per ear was highest in the OMWW treatment, which indicated an 18.0% increase compared to the control (p < 0.05). Conversely, drought stress decreased the number of grains per ear, a drop of 27.1% (p < 0.05). The greatest 1000-grain weight was found in the OMWW-treated plants, which was non-significantly greater than in control plants (p ≥ 0.05). The drought stress condition resulted in a lower weight by 16.8% compared to the control (p < 0.05). OMWW combined with stress further reduced weight, marking an 18.2% decrease relative to the control (p < 0.05).

Soil physicochemical characteristics

The analysis of soil physicochemical characteristics revealed significant effects of applied treatments on some main parameters (Table 1). In this regard, the OMWW treatment showed the highest EC value, which was significantly higher than control treatments (+ 84.8%) (p < 0.05). Similarly, drought stress treatment recorded a 64.8% increase compared to control (p < 0.05). The OMWW-containing treatments significantly enhanced organic matter content compared to control and stress treatments (p < 0.05). Accordingly, the OMWW + Stress treatment showed improved organic matter at 3.25 g kg^-1, leading to a 49.8% increase relative to the control (p < 0.05). The total P level in soil was highest in the OMWW + stress-treated plants, resulting in a remarkable 101% increase in comparison with the control (p < 0.05). In comparison, the total P for the OMWW treatment yielded an increase of 88.4% from the control. Moreover, the OMWW treatment demonstrated the highest K concentration, indicating an increase of 88.1% over the control. The combined treatment of OMWW + stress yielded an 81.2% increase compared to the control (p < 0.05). OMWW + stress treatment resulted in the highest Ca level in the soil, which showed an 88.9% increase in comparison to the control soil. The OMWW treatment contributed 22.9 mg Ca Kg^-1 in the soil, reflecting a 76.6% increase from the control.

Table 1.

Soil physicochemical characteristics in response to drought stress and Olive mill wastewater treatment (OMWW).

Parameter	Control	Stress	OMWW	OMWW + Stress
E.C. (dS m− 1)	1.65 ± 0.24 b	2.72 ± 0.35 a	3.11 ± 0.30 a	2.74 ± 0.21 a
pH	6.97 ± 0.45 a	6.60 ± 0.55 a	6.71 ± 0.73 a	6.45 ± 0.21 a
CaCO3 (g Kg− 1)	33.95 ± 4.21 b	37.26 ± 3.77 b	52.32 ± 4.11 a	51.62 ± 5.05 a
Organic matter (g Kg− 1)	2.17 ± 0.15 b	2.35 ± 0.21 b	3.86 ± 0.35 a	3.25 ± 0.41 a
Total N (mg Kg− 1 soil)	274.91 ± 31.12 a	256.00 ± 27.78 a	154.23 ± 21.13 b	254.74 ± 19.93 a
NH4-N	6.87 ± 0.45 b	11.32 ± 1.01 a	10.69 ± 1.43 a	12.37 ± 1.87 a
Total P (mg Kg− 1 soil)	125.09 ± 10.41 b	138.86 ± 15.19 b	235.50 ± 21.25 a	251.44 ± 26.32 a
K (mg Kg− 1 soil)	114.59 ± 10.36 b	103.49 ± 9.71 b	215.41 ± 14.19 a	207.57 ± 28.02 a
Ca (mg Kg− 1 soil)	12.97 ± 2.03 b	22.71 ± 1.78 a	22.88 ± 1.99 a	24.49 ± 2.24 a
Mg (mg Kg− 1 soil)	6.65 ± 0.54 b	11.37 ± 0.87 a	13.22 ± 1.41 a	11.03 ± 1.33 a
Fe (mg Kg− 1 soil)	3.14 ± 0.17 b	4.86 ± 0.54 a	5.04 ± 0.35 a	5.24 ± 0.55 a

At a 5% probability level, the Tukey HSD test reveals that soil variables sharing the same letter(s) were not significantly different.

Sugar metabolism

The results also indicated significant changes in some sugar content and enzyme activity in wheat grains subjected to drought stress and OMWW treatment (Table 2). Under OMWW treatment, total sugar content remained unchanged compared to the control, while under combined OMWW + stress conditions, it reached a 24.6% increase over the control (p < 0.05). Notably, the starch content in the grains showed a significant increase of 29.5% in OMWW-treated plants compared to control and a 25.9% increase compared to stressed plants (p < 0.05), whereas, under OMWW + stress conditions, starch levels were elevated non-significantly relative to stressed and control plants (p ≥ 0.05). Fructose concentrations showed a significant rise of 46.5% in OMWW + stress-treated plants compared to control, while glucose levels increased by 27.2% under OMWW + stress compared to the control (p < 0.05). Additionally, invertase and sucrose phosphate synthase activity remained unchanged in response to the treatments (p ≥ 0.05). Amylase activity peaked under OMWW + stress, showing a remarkable increase of 39.7% and 51.7% over the control and stress treatments, respectively (p < 0.05).

Table 2.

Sugar content and activity of enzymes involved in their metabolism in wheat grains under drought stress and Olive mill wastewater treatment.

Parameter	Control	Stress	OMWW	OMWW + Stress
Total Sugar	4.85 ± 0.55 b	5.61 ± 0.46 ab	5.14 ± 0.43 ab	6.05 ± 0.45 a
Total soluble sugar	3.02 ± 0.29 a	3.51 ± 0.35 a	2.89 ± 0.41 a	2.97 ± 0.33 a
Fructose	0.43 ± 0.05 b	0.51 ± 0.06 ab	0.50 ± 0.04 b	0.63 ± 0.06 a
Glucose	0.29 ± 0.04 a	0.32 ± 0.03 a	0.30 ± 0.04 a	0.37 ± 0.04 a
Sucrose	1.05 ± 0.15 a	1.21 ± 0.21 a	1.09 ± 0.15 a	1.04 ± 0.18 a
Starch	505.81 ± 35.21 b	519.84 ± 44.12 ab	654.84 ± 0.65 a	546.69 ± 0.61 ab
Invertase	1.44 ± 0.16 a	1.38 ± 0.18 a	1.32 ± 0.19 a	1.24 ± 0.21 a
Sucrose phosphte synthase	0.29 ± 0.03 a	0.33 ± 0.04 a	0.30 ± 0.04 a	0.37 ± 0.05 a
Amylase	0.63 ± 0.08 b	0.58 ± 0.08 b	0.69 ± 0.09 ab	0.88 ± 0.10 a
Starch Synthase	30.96 ± 2.88 b	32.44 ± 3.21 b	39.00 ± 3.02 a	42.75 ± 3.67 a

At a 5% probability level, the Tukey HSD test reveals that grain parameters sharing the same letter(s) is not significantly different.

Mineral content in grains

In the analysis of mineral content in wheat grains, OMWW treatment significantly enhanced several mineral contents compared to control and stress treatments (Table 3).Notably, phosphorus content reached its highest level under OMWW treatment, representing an approximate 31% increase over the control plants subjected to drought stress (p < 0.05). Similarly, potassium levels in the grains exhibited a significant rise in OMWW-treated plants compared to the stressed and control groups, indicating enhanced mineral uptake (p < 0.05). Calcium content also displayed significant differences, with levels significantly higher in the drought stress and OMWW treatments, specifically marking a 41.4% and 67.7% increase over the control plants, respectively (p < 0.05). Furthermore, sodium and manganese concentrations were significantly elevated in treatments that included OMWW, reflecting its role in enhancing mineral availability (p < 0.05). Iron (Fe) content was also markedly increased in OMWW-treated plants under both drought stress and non-stress conditions compared to control and stressed plants, reinforcing the effectiveness of OMWW in improving the nutritional profile of wheat grains (p < 0.05). These findings suggest that OMWW can positively impact the mineral composition of wheat.

Table 3.

Mineral content in wheat grains under drought stress and Olive mill wastewater treatment.

Parameter	Control	Stress	OMWW	OMWW + Stress
P (mg g− 1)	4.43 ± 0.44 a	3.68 ± 0.59 b	4.87 ± 0.51 a	4.82 ± 0.56 a
S (mg g− 1)	0.98 ± 0.13 a	0.96 ± 0.16 a	1.13 ± 0.14 a	1.26 ± 0.21 a
K (mg g− 1)	7.90 ± 0.45 b	7.42 ± 0.57 b	10.35 ± 0.96 a	10.44 ± 0.55 a
Mg (mg g− 1)	1.15 ± 0.24 a	1.22 ± 0.14 a	1.26 ± 0.18 a	1.33 ± 0.19 a
Ca (µg g− 1)	70.27 ± 8.88 b	99.35 ± 10.12 a	117.82 ± 15.12 a	102.98 ± 11.16 a
Na (µg g− 1)	9.93 ± 0.89 b	9.60 ± 0.76 b	13.09 ± 0.96 a	13.52 ± 1.12 a
Zn (µg g− 1)	30.49 ± 4.11 a	27.70 ± 3.79 a	33.42 ± 3.34 a	35.49 ± 5.02 a
Fe (µg g− 1)	31.02 ± 1.88 b	31.61 ± 3.12 b	48.60 ± 3.35 a	49.18 ± 2.22 a
Mn (µg g− 1)	4.02 ± 0.27 b	2.76 ± 0.31 c	6.35 ± 0.44 a	6.75 ± 0.49 a

At a 5% probability level, the Tukey HSD test reveals that sharing the same letter(s) is not significantly different.

Antioxidant molecules and enzymes

The analysis of antioxidant capacity and enzyme activity in wheat grains under drought stress and OMWW treatment, significant increases were observed in several parameters (Table 4). The total antioxidant capacity was significantly elevated by 60.5% in OMWW-treated plants compared to the control (p < 0.05). Polyphenol content increased by 83.2% in the OMWW + stress treatment over the control (p < 0.05), while flavonoid levels showed a significant rise of 86.2% in OMWW + stress compared to the control (p < 0.05). ASC levels rose by 62.1% in OMWW-treated plants, and GSH content increased by 82.4% in response to the OMWW + stress treatment compared to the control (p < 0.05). Among antioxidant enzymes, CAT activity reached the highest activity under OMWW + stress, representing a 44.6% increase over the control plants (p < 0.05). SOD activity also increased significantly by 46.6% under OMWW + stress compared to the control (p < 0.05). These results highlight the enhanced antioxidant capacity and enzyme activities facilitated by OMWW treatment, particularly under stress conditions. In the analysis of tocopherol content in wheat grains, alpha-tocopherol showed a significant increase of 44.4% and 27.4% in the OMWW + stress treatment compared to the control and stressed plants, respectively (p < 0.05) (Figure. 2). In contrast, beta-tocopherol and gamma-tocopherol did not show significant differences among treatments, remaining relatively stable across all conditions (p ≥ 0.05).

Table 4.

Total antioxidant capacity (µmol trolex g^-1 FW), antioxidant molecules content (µmol g^-1 FW), and the activity of antioxidant enzymes (mol min^-1 mg^-1 protein) in wheat grains under drought stress and Olive mill wastewater treatment (OMWW).

Parameter	Control	Stress	OMWW	OMWW + Stress
Total antioxidant capacity	821.14 ± 100.13 b	981.49 ± 90.19 b	1317.64 ± 100.20 a	1288.23 ± 85. 65 a
Polyphenols	50.88 ± 6.11 c	61.95 ± 6.66 bc	74.76 ± 8.19 b	93.08 ± 6.27 a
Flavonoids	2.46 ± 0.31 b	2.96 ± 0.35 b	3.96 ± 0.44 a	4.58 ± 0.41 a
Ascorbate	2.14 ± 0.32 b	2.70 ± 0.39 ab	3.47 ± 0.35 a	3.25 ± 0.29 a
Glutathione	0.91 ± 0.15 b	1.02 ± 0.09 b	1.31 ± 0.31 ab	1.66 ± 0.26 a
Peroxidase	47.21 ± 6.11 b	54.84 ± 5.59 ab	68.02 ± 8.05 a	65.58 ± 7.19 a
Catalase	159.11 ± 21.08 b	180.08 ± 23.19 ab	198.61 ± 26.23 ab	230.11 ± 31.17 a
Superoxide Dismutase	1688.67 ± 150.34 b	1882.18 ± 169.93 b	1528.45 ± 200.49 b	2470.10 ± 190.74 a
ascorbate peroxidase	15.24 ± 2.01 b	17.88 ± 2.89 ab	22.13 ± 2.23 a	22.53 ± 3.11 a
Dehydroascorbate reductase	5.00 ± 0.83 b	5.50 ± 0.55 b	6.70 ± 0.98 ab	7.56 ± 0.76 a
monodehydroascorbate reductase	4.60 ± 0.45 b	5.10 ± 0.41 b	5.40 ± 0.71 b	7.08 ± 0.46 a
Glutathione reductase	67.28 ± 5.87 b	73.17 ± 7.19 b	99.96 ± 5.47 a	100.38 ± 11.10 a
Glutathione peroxidase	27.09 ± 2.18 b	31.25 ± 2.77 b	39.04 ± 1.89 a	40.48 ± 4.12 a
Glutaredoxins	12.57 ± 1.87 b	15.69 ± 1.76 ab	18.59 ± 2.04 a	20.35 ± 2.77 a
Thioredoxin	1.20 ± 0.09 b	1.35 ± 0.25 b	1.82 ± 0.14 a	2.10 ± 0.19 a
Peroxiredoxins	39.92 ± 3.11 b	55.43 ± 5.53 a	58.09 ± 2.19 a	58.06 ± 3.67 a

At a 5% probability level, the Tukey HSD test reveals that sharing the same letter(s) is not significantly different.

Grain compositions

Analyzing grain composition and nutritional values revealed significant enhancements in plants treated with OMWW and those subjected to drought stress (Table 5). Notably, crude protein content increased by 48.7% in the OMWW + stress treatment compared to the control (p < 0.05). Lipid content demonstrated a 29.4 rise in the OMWW + stress group relative to the control plants (p < 0.05). Additionally, NDF and ADF were elevated by 51.7% and 37.3%, respectively, in the OMWW + stress treatment compared to the control (p < 0.05). Furthermore, the tannin levels significantly increased by 51.7% in the OMWW + stress group compared to the control (p < 0.05).

Table 5.

Grain composition and nutritional values in plants under drought stress and Olive mill wastewater.

Parameter	Control	Stress	OMWW	OMWW + Stress
Crude protein	12.18 ± 2.00 b	15.27 ± 1.89 ab	16.14 ± 2.25 ab	18.15 ± 0.23 a
Ash	1.86 ± 0.15 a	1.92 ± 0.19 a	2.12 ± 0.21 a	2.12 ± 0.16 a
Lipids	3.86 ± 0.51 b	4.83 ± 0.54 ab	4.52 ± 0.47 ab	5.00 ± 0.34 a
NDF	95.19 ± 6.76 c	135.72 ± 14.65 a	131.90 ± 12.78 a	144.35 ± 16.22 a
ADF	42.79 ± 4.45 b	48.46 ± 4.77 ab	51.68 ± 6.01 ab	58.74 ± 6.12 a
ADL	1.03 ± 0.12 b	1.22 ± 0.29 ab	1.25 ± 0.21 ab	1.55 ± 0.21 a
Tannins	0.29 ± 0.04 b	0.35 ± 0.04 ab	0.36 ± 0.04 ab	0.44 ± 0.05 a
Alkaloids	1.78 ± 0.15 c	3.11 ± 0.42 b	2.55 ± 0.33 bc	4.56 ± 0.29 a

At a 5% probability level, the Tukey HSD test reveals that parameters sharing the same letter(s) is not significantly different. ADF: Acid detergent fiber; NDF: Neutral detergent fiber; ADL: Acid detergent lignin.

Profiles of amino acids, fatty acids, and phenolic acids

In this study, the profiles of amino acids, fatty acids, and phenolic acids in wheat grains were significantly influenced by OMWW treatment under drought stress conditions. Moreover, the hierarchical clustering analysis showed distinct shifts in amino acids, fatty acids, and phenolics compositions in plants in response to OMWW treatment and drought stress conditions, in which the OMWW-containing treatments (OMWW and OMWW + stress) were clustered into a different group compared to no OMWW treatments (stress and control treatments) (Figs. 3, 4, and 5). Among phenolic acids, p-Coumaric acid was the most abundant and exhibited the highest increase of 53.7% in the OMWW treatment compared to the control treatment (Fig. 5). In the amino acid analysis, proline, ornithine, and arginine were the most abundant amino acids in the grains, which showed an impressive increase of 51.7%, 56.3%, and 50.0% in the OMWW + stress treatment compared to the control. Moreover, the oleic acid (octadecenoic; C18:1) and linoleic acid (octadecadienoic; C18:2) were the most abundant fatty acids observed in the grains, which were significantly higher in OMWW + Stress treatment compared to control. In contrast, palmitic acid (hexadecanoic; C16:0) was the most abundant fatty acid that showed a decrease in content under stress and OMWW treatments (Fig. 4).

Fig. 3.

A heat map created through hierarchical clustering analysis (employing Ward’s method) displayed the changes in the content of various amino acids that were differentially altered in plants under drought stress and olive mill wastewater treatment (OMWW). The color bars at the bottom of the panel, ranging from 0 to 6.5, represent the values of the parameters.

Fig. 4.

A heat map produced through hierarchical clustering analysis (using Ward’s method) revealed the variations in the content of differentially altered fatty acids in plants under drought stress and olive mill wastewater treatment. The color bars at the bottom of the panel, ranging from 0 to 25, represent the values of the parameters.

Fig. 5.

A heat map constructed using hierarchical clustering analysis (employing Ward’s method) displayed the variations in the content of differentially altered phenolic acids in plants under drought stress and olive mill wastewater treatment. The color bars at the bottom of the panel, ranging from 0 to 1.7, represent the values of the parameters.

Fig. 1.

Plant yield and yield components in response to drought stress and olive mill wastewater. At a 5% probability level, the Tukey HSD test reveals that sharing the same letter(s) is not significantly different.

Fig. 2.

Tocopherols content (ng g^− 1 FW) in wheat grains in response to drought stress and olive mill wastewater treatment. At a 5% probability level, the Tukey HSD test reveals that sharing the same letter(s) is not significantly different.

Organic acids

Assessment of organic acid content in wheat grains revealed significant increases in several parameters following treatment with OMWW and drought stress (Table 6). Succinate levels rose by about 57–63% in the OMWW-containing treatments compared to the control and stressed plants (p < 0.05). A similar finding was observed in oxalate content in the grains in response to the OMWW and OMWW + stress treatments, with an increase of approx. 95–106% compared to the stress and control treatments (p < 0.05). Moreover, citrate content was significantly higher in the OMWW and OMWW + stress treatments over the control and stress treatments (p < 0.05). Additionally, lactate levels in the OMWW + stress treatment reached the highest value, representing a 33.1% increase compared to the stress treatment (p < 0.05).

Table 6.

Organic acid content in wheat grains in response to drought stress and Olive mill wastewater treatment (OMWW).

Parameter	Control	Stress	OMWW	OMWW + Stress
Succinate	305.07 ± 19.43 b	298.87 ± 24.45 b	478.17 ± 35.56 a	488.20 ± 20.88 a
Malate	96.88 ± 9.75 a	89.52 ± 10.00 a	93.72 ± 8.87 a	110.05 ± 14.01 a
Citrate	157.79 ± 21.26 b	191.09 ± 18.94 b	249.29 ± 31.90 a	282.04 ± 30.83 a
Lactate	155.93 ± 14.87 b	156.67 ± 9.77 b	193.45 ± 15.92 a	208.57 ± 21.63 a
Trans-aconitic	32.22 ± 3.11 a	25.84 ± 4.21 a	27.00 ± 3.79 a	32.34 ± 4.01 a
Oxalate	57.13 ± 3.33 b	54.80 ± 4.19 b	111.52 ± 9.98 a	113.04 ± 11.62 a

At a 5% probability level, the Tukey HSD test reveals that parameters sharing the same letter(s) is not significantly different.

Polyamines metabolism

The results also indicated that OMWW treatment significantly improved the content of some key polyamines and related enzymes in wheat grains under stress conditions (Table 7). In this regard, diaminopropane content in the grains increased in response to OMWW + stress treatment by 36.5% and 52.4% compared to the control and stressed plants, respectively (p < 0.05). Put levels rose by 95–101% in the OMWW-containing treatments compared to the stress treatment (p < 0.05). Additionally, ODC and AdoMetDC activity demonstrated a significant increase of 94.8% and 83.7% in the OMWW + stress treatment compared to the control, respectively (p < 0.05). For PAO activity, the OMWW treatment also showed the highest value, representing a 32.5% increase compared to the stress treatment (p < 0.05). Moreover, DAO activity was highest in the stress treatment but saw a significant decrease of 46.2% in the OMWW + stress treatment (p < 0.05).

Table 7.

Polyamines content and the activity of enzymes involved in their metabolism in wheat grains under drought stress and Olive mill wastewater treatment.

Parameter	Control	Stress	OMWW	OMWW + Stress
Diaminopropane	1.15 ± 0.09 b	1.03 ± 0.18 b	1.19 ± 0.07 b	1.57 ± 0.11 a
Putrescine	26.07 ± 3.04 a	15.09 ± 1.70 b	29.37 ± 3.11 a	30.45 ± 2.89 a
Cadaverine	4.20 ± 0.32 a	1.75 ± 0.21 b	4.19 ± 0.38 a	1.91 ± 0.17 b
Spermidine	43.66 ± 4.11 a	52.81 ± 6.14 a	43.21 ± 5.34 a	51.43 ± 5.18 a
Spermine	61.21 ± 7.89 a	73.24 ± 8.11 a	60.93 ± 7.56 a	59.39 ± 7.43 a
Agmatine	1.16 ± 0.11 a	0.50 ± 0.05 b	0.55 ± 0.04 b	0.48 ± 0.05 b
ODC	300.45 ± 31.54 c	459.61 ± 40.66 b	468.92 ± 30.45 b	584.62 ± 41.47 a
AdoMetDC	323.23 ± 28.54 c	417.78 ± 22.45 b	452.25 ± 43.67 b	594.37 ± 39.67 a
SPMS	492.12 ± 56.87 a	215.05 ± 34.10 b	427.82 ± 45.44 a	393.52 ± 50.02 a
SPDS	455.54 ± 45.66 a	277.75 ± 35.76 b	513.50 ± 46.98 a	445.26 ± 52.22 a
ADC	293.81 ± 35.18 b	344.13 ± 29.36 b	449.36 ± 40.43 a	479.19 ± 37.48 a
PAO	316.19 ± 22.19 b	287.16 ± 25.66 b	418.65 ± 30.48 a	269.95 ± 39.56 b
DAO	418.95 ± 45.99 a	542.62 ± 53.49 a	502.33 ± 50.01 a	288.54 ± 33.45 b

AdoMetDC: S-adenosyl-L-methionine decarboxylase; ADC: Arginine decarboxylase; DAO: Diamino oxidases; ODC: Ornithine decarboxylase; PAO: Polyamine oxidase; Spm: Spermine; Spd: Spermidine; SPMS: Spm synthase; SPDS: Spd synthase.

Discussion

The results detail significant biochemical and compositional changes in wheat grains resulting from OMWW treatment, particularly under drought stress conditions. The findings indicate that OMWW improves plant resilience to stress and enhances the grains’ nutritional and functional characteristics. The results concerning agronomic traits in the context of drought stress and OMWW treatment reveal crucial insights into how these factors influence plant productivity. Specifically, the findings indicate that while OMWW treatment enhances several yield components, its effects are mitigated under drought stress conditions. Under drought stress, the grain yield in OMWW-treated plants was higher than in untreated plants, which reflects an essential aspect of OMWW’s potential role as a beneficial soil amendment. These improvements were accompanied by increasing the levels of some key nutrients in the grain of stressed plants, such as P, K, Fe, and Mn. This observation underscores the utility of OMWW in improving wheat grain production and nutrient uptake. In addition to mitigating these stress-induced reductions, OMWW treatment facilitated critical adjustments in plant metabolism, promoting tolerance to abiotic stress. These findings suggest that OMWW’s ability to influence multiple physiological pathways could extend its applicability to other cereal crops or agricultural systems under similar environmental constraints¹⁵. This potential for broader applicability highlights the value of OMWW as a versatile tool in sustainable agriculture.

When examining the impact of drought stress alone, a significant decrease in grain yield and agronomic traits was recorded compared to the control. This decline showed the detrimental effects of drought stress on wheat plants, reflecting how stress conditions can drastically impair plant growth and productivity. Drought stress significantly reduces wheat grain yield by limiting water availability, impairing photosynthesis, hindering nutrient uptake, disrupting metabolism, and causing physiological damage from reactive oxygen species, all of which collectively diminish the quantity and quality of the grains¹⁶. However, the results from this study demonstrate that OMWW applications can partially mitigate these negative impacts. By improving soil properties, nutrient availability, and metabolic responses, OMWW-treated plants under drought stress exhibited higher grain yield and enhanced biochemical traits than untreated plants. This highlights the potential of OMWW as an effective amendment for counteracting the physiological and metabolic disruptions caused by drought stress.

A notable finding from this research is the significant increase in amylase activity observed in plants treated with OMWW under stress conditions compared to the control group. This increase in amylase activity indicates a heightened activation of starch breakdown processes, which are vital for mobilizing energy during challenging circumstances. This observation aligns with previous studies highlighting amylase’s key role in carbohydrate hydrolysis, providing essential energy sources for plant growth and resilience under unfavorable environmental conditions¹⁷. The obvious elevation in sucrose concentrations under OMWW treatment, combined with stable invertase and SPS activities, supports the hypothesis that OMWW alters carbohydrate metabolism favorably to enhance vigor and stress tolerance. In this context, the observed changes in lipid metabolism indicate a targeted response to oxidative stress, suggesting that OMWW treatment not only improves physical resilience but also enhances signaling pathways essential for adaptive mechanisms under drought conditions. Future research could explore how these metabolic adjustments vary among different plant species to assess OMWW’s broader utility. Furthermore, the modifications observed in lipid content and fatty acid composition deserve particular attention, as the levels in grains increased following the OMWW + stress treatment compared to control plants. Lipids are essential for forming cellular membranes and preserving membrane integrity during stress, making this enhancement vital for maintaining cellular structure and function¹⁸. The rise in oleic acid (C18:1) and linoleic acid (C18:2) is especially significant, as these unsaturated fatty acids improve membrane fluidity. Specifically, an increase in oleic acid enhances the plant’s ability to cope with oxidative stress¹⁹, while linoleic acid acts as a precursor to crucial signaling molecules that help regulate stress responses.

The study also highlights significant increases in soluble fiber levels, particularly NDF and ADF, in the OMWW + stress treatment. This fiber accumulation not only enhances the structural strength of cell walls but may also act as a protective mechanism against pest infestations and disease²⁰. Additionally, these fibers can improve the digestibility and overall nutritional value of the grains, offering benefits to consumers. Moreover, these findings underscore the dual benefits of OMWW treatment in improving crop resilience and food quality. This proposes OMWW as a promising candidate for integrated agricultural systems to meet the growing demand for nutritious food under challenging climatic conditions.

Moreover, the significant increase in crude protein content under OMWW + stress versus control cannot be overlooked. This phenomenon likely results from a combination of enhanced N assimilation and better overall plant health fostered by OMWW treatment. Protein synthesis typically ramps up under stress as plants allocate resources toward critical cellular functions and repair mechanisms²¹. This can be related to the increase in the concentration of some key amino acids involved in N metabolism in the grains of the present study, such as proline and arginine²¹.

Moreover, the significant increases in some amino acids (e.g., proline, ornithine, and arginine) in the OMWW + stress treatment can be partially attributed to the polyamine pathway’s interaction with amino acid metabolism. These amino acids not only serve as precursors to polyamines but also play essential roles in managing drought balance, cellular signaling, and responding to stress²². The observed increase in polyamines and related enzyme activity further supports the role of OMWW as a biostimulant, with its effects on polyamine synthesis providing a mechanistic basis for the observed enhanced stress tolerance²³. These findings suggest the potential for OMWW-based formulations to be tailored for specific stress management applications in agriculture. These enzymes facilitate the conversion of amino acids into polyamines, thereby promoting their accumulation²⁴. The enhanced enzyme activity in this treatment indicates that this enzymatic regulation serves as a strategic adaptive mechanism, helping plants manage drought stress by raising polyamine levels, which contribute to stabilizing cellular structures and reducing stress-related damage²⁴. Moreover, the peak PAO activity observed with the OMWW treatment points to a complex regulatory mechanism, suggesting that polyamine degradation may be carefully managed in the context of drought stress. While PAO is tasked with the breakdown of polyamines, maintaining a balance between synthesis and degradation becomes particularly critical under stressful conditions. The decrease in DAO activity observed in the OMWW + stress treatment aligns with the idea that OMWW helps create a more conducive metabolic environment for polyamine accumulation, counteracting the typical degradation pathways that can be activated during stress²⁵.This study highlights the elevated presence of antioxidant molecules and enzymes, which serve as a mechanism by which plants combat oxidative damage caused by drought stress. Notably, certain antioxidant enzymes showed a marked increase in activity when subjected to stress, particularly in plants treated with OMWW. Previous research has established a connection between the detoxification of excess reactive oxygen species (ROS) in plant cells and the enhancement of antioxidant protection mechanisms in those treated with OMWW²⁶. However, it is important to carefully consider antioxidant enzymes’ different responses to stress and OMWW treatments. The interactions between these antioxidant pathways and other signaling molecules and metabolites during oxidative stress are still poorly understood²⁷. Moreover, it is important to recognize the susceptibility of components within the ASC/GSH cycle and other antioxidant pathways to oxidative stress, as this could impact their antioxidant effectiveness under stress, even though they play a crucial role in safeguarding cellular integrity from oxidative damage^27,28.

We investigated antioxidant molecules involved in various antioxidant pathways to determine if specific treatments could improve the antioxidant capacity of plants. Our results clarify that OMWW treatments significantly boost the levels of key antioxidant molecules such as alpha-tocopherol, polyphenols, flavonoids, and total antioxidant capacity in plants. These metabolites are vital for helping plants acclimate to stress conditions^1,28, particularly in safeguarding the photosynthetic apparatus²⁹. The increase in antioxidant molecules noted in this study, particularly in plants treated with OMWW, appears to be a critical strategy for preservation against drought stress. The potential of olive waste to enhance plant resilience to oxidative stress has been linked to its elevated levels of certain metabolites, particularly phenolic compounds like p-coumaric acid, syringic acid, and gallic acid³⁰.

Nevertheless, the findings of the present research should be interpreted with caution, as studies have indicated that OMWW can pose substantial long-term environmental and phytotoxicity risks, negatively impacting plant production and soil health³¹. High levels of organic matter, phenolic compounds, and other toxic constituents in OMWW can lead to soil acidification, disrupt microbial communities, and reduce soil fertility^31,32. The accumulation of these substances may inhibit seed germination and root development, ultimately diminishing crop yield and plant vitality³³. Therefore, effective management strategies are crucial to mitigate these negative impacts and promote sustainable agricultural practices. Future research should prioritize long-term field studies to assess the cumulative effects of OMWW application on soil health and crop productivity across diverse agroecological zones. Additionally, exploring OMWW’s synergistic potential with other organic amendments may yield optimized formulations for sustainable agriculture.

Materials and methods

Plant treatment

Eight healthy seeds of wheat (variety Giza 119) were sterilized with a 1% (v/v) NaClO solution for 10 min. After sterilization, the seeds were planted in a potting mix of Tref EGO substrates. The experiment was conducted in 2023 at the Botany and Microbiology Department, College of Science, King Saud University, using pots measuring 15 cm × 15 cm. Each pot was filled with a mixture of loamy soil and organic compost in a 1:1 (v/v) ratio, maintaining a humidity level of 300 mg of water per gram of dry soil. The plants were then grown in a chamber with a controlled condition with the following conditions: 20 °C air temperature, 14/10 h day/night photoperiod, and photosynthetically active radiation of 150 µmol m^− 2 s^− 1 for the tillering stage and 550 µmol m^− 2 s^− 1 for the harvesting stage.

The experimental design was based on a two-way ANOVA analysis, with stress conditions (control vs. drought stress) as the first factor and the type of irrigation material (OMWW vs. water) as the second factor. Several concentrations of OMW solutions (12.5, 25, 75, 150, 300, and 600 ml L⁻¹.) were tested. Based on the experimental outcomes, the concentration of 75 ml L⁻¹ was selected for further analysis, as it improved wheat growth under drought stress without causing adverse on plants grown under control conditions. Drought stress treatment in pots was implemented by withholding irrigation until the soil field capacity (FC) dropped to 40%, indicating severe stress conditions. Several field FCs were tested in the previous experiments, and 40% FC was selected as the severe stress condition because it induced about a 50% reduction in plant growth without causing plant mortality. Stress was imposed at the tillering stage (30 days after sowing). Control pots were kept at 75% FC throughout the experiment. In detail, FC of the soil used in the experiments was determined using a pressure membrane chamber apparatus, as described by Gugino et al.³⁴. For 75% FC, approximately 7.8 L of water were required in each pot throughout the growth period, which was distributed effectively and consistently according to the irrigation plan. Conversely, drought stress treatment at 40% FC was implemented by manually adjusting the water volume based on the calculated field capacity. Specifically, approximately 4.2 L of water were required for each pot at 40% FC. For the pots subjected to OMWW treatment, OMWW was applied instead of water in the same amount. Wheat grains were harvested 155 days after sowing (DAS) (full maturation of the grains). The samples of OMWW were collected, and different physical and chemical properties of raw and diluted OMWW are presented in Table 8. The wastewater samples were collected in plastic bottles and subsequently used for irrigation.

Table 8.

 Physiochemical and antioxidant properties of Olive mill wastewater.

Physico-chemical characterization	Raw OMWW	diluted-OMWW
Dry matter (%)	17.99 ± 1.65	11.78 ± 1.11
pH	5.97 ± 0.46	5.89 ± 0.39
Electric conductivity (dS m− 1 )	10.42 ± 0.87	1.25 ± 0.11
Chemical oxygen demand (g L− 1 )	16.61 ± 1.34	12.82 ± 1.00
Biochemical oxygen demand (g L− 1)	70.14 ± 5.67	52.98 ± 4.17
Organic matter (g L− 1 )	19.17 ± 2.23	11.73 ± 1.45
Minerals (g L− 1 )
Nitrogen	2.46 ± 0.23	1.45 ± 0.18
Phosphorus	23.11 ± 1.78	10.31 ± 1.09
Potassium	4.66 ± 0.46	2.35 ± 0.31
Calcium	0.93 ± 0.08	0.52 ± 0.06
Magnesium	0.50 ± 0.07	0.28 ± 0.04
Sodium	1.78 ± 0.54	0.99 ± 0.07
Chloride	1.25 ± 0.16	0.70 ± 0.10
Iron	0.72 ± 0.05	0.41 ± 0.05
Zinc	0.18 ± 0.01	0.10 ± 0.02
Cadmium	0.07 ± 0.00	0.04 ± 0.00
Lead	0.02 ± 0.00	0.01 ± 0.00
Copper	0.06 ± 0.00	0.03 ± 0.00
Nickel	0.02 ± 0.00	0.01 ± 0.00
Chromium	0.02 ± 0.00	0.01 ± 0.00
Arsenic	0.002 ± 0.00	0.001 ± 0.00
Mercury	0.0001 ± 0.00	0.0000 ± 0.00
Antioxidant
Antioxidant Activity (FRAP)	63.80 ± 8.11	42.14 ± 6.35
Antioxidant Activity DPPH (%)	89.50 ± 10.44	52.70 ± 7.34
Phenols and flavones (g L− 1 )
Total phenols	4.32 ± 0.34	3.30 ± 0.23
Total Flavonoids	3.45 ± 0.25	2.07 ± 0.27
Caffeic acid	0.03 ± 0.00	0.01 ± 0.00
Ferulic ACID	0.16 ± 0.02	0.09 ± 0.01
Protocatechuic acid	0.31 ± 0.04	0.17 ± 0.03
Catechin	0.26 ± 0.04	0.15 ± 0.02
Galic acid	0.18 ± 0.02	0.10 ± 0.02
p-Coumaric acid	0.55 ± 0.07	0.34 ± 0.04
Resorcinol	0.05 ± 0.00	0.38 ± 0.04
Chlorogenic acid	0.33 ± 0.06	0.21 ± 0.03
Syringic acid	0.16 ± 0.02	0.08 ± 0.01
Quercetin	0.08 ± 0.01	0.45 ± 0.03
Quercetrin	0.13 ± 0.03	0.10 ± 0.02
Luteolin	0.18 ± 0.02	0.10 ± 0.01
Apigenin	0.23 ± 0.03	0.17 ± 0.02
Isoquercetrin	0.56 ± 0.04	0.34 ± 0.04
Rutin	0.06 ± 0.00	0.04 ± 0.00
Ellagic acid	0.02 ± 0.00	0.14 ± 0.02
Velutin	0.02 ± 0.00	0.08 ± 0.01
Naringenin	0.08 ± 0.01	0.07 ± 0.01
Genistein	0.14 ± 0.02	0.08 ± 0.01
Daidzein	0.20 ± 0.03	0.09 ± 0.01
Fisetin	0.26 ± 0.04	0.17 ± 0.03
O-hydroxydaidzein	0.05 ± 0.00	0.03 ± 0.00

All assessments were conducted following established protocols for analyzing water and wastewater.

Plant traits assessment

At the harvest stage (155 DAS), plants were collected to evaluate the different growth and yield parameters, including grain yield per plant, ear length, number of grains per ear, spikelet per ear, and grain weight.

Sample preparation and analysis conditions

For further analyses, the harvested grains were carefully cleaned to remove any debris and then placed at room temperature to a constant weight. Dried samples were ground using a mortar and pestle to achieve a fine powder, which was stored in airtight containers at -20 °C until analysis.

Determination of mineral concentration

The accumulation of minerals in both soil and plant samples was analyzed using total-reflection X-ray fluorescence spectrometry (TXRF; Bruker Nano GmbH, Berlin, Germany), inductively coupled plasma optical emission spectroscopy (ICP-OES), and the Kjeldahl method^35,36. In detail, For TXRF analysis, approximately 1 gram of dried soil or plant sample was mixed with a binding agent and placed onto a sample holder, followed by measurement under standardized conditions to quantify trace element concentrations. For ICP-OES, soil and plant samples (0.5 g) were digested in a microwave with a 3:1 v/v mixture of concentrated nitric acid and hydrochloric acid, then diluted with deionized water for mineral analysis, focusing on key nutrients such as phosphorus, potassium, and calcium. The total nitrogen content in plant samples was determined using the Kjeldahl method, which involved acid digestion followed by neutralization and distillation to quantify ammonia released, providing insights into the nitrogen content.

Nutritional components of grains

The concentration of total polyphenols and flavonoids in grain samples was quantified according to the methodologies outlined by Zhang et al.³⁷ and Chang et al.³⁸, utilizing a spectrophotometric approach where extracts from the grains were obtained through solvent extraction with 80% methanol and subsequently assayed for absorbance at specific wavelengths. The total protein content was determined using the color intensity proportional to the protein amount in the sample, and the absorbance was detected at 562 nm³⁹. Crude fiber content was assessed following the AOAC guidelines⁴⁰, where dried samples were treated with dilute sulfuric acid and sodium hydroxide, and the insoluble residue was quantified gravimetrically. Tannin content was analyzed via solvent extraction with HCl (1% v/v) in methanol, treated with vanillin-HCl, and measured at 500 nm⁴¹. The ash content was determined according to the method established by Czaja et al.⁴², involving incineration of the samples at high temperatures to measure inorganic residue. Alkaloids were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods as described by Babič et al.⁴³, enabling sensitive detection of diverse alkaloid compounds. Finally, saponin content was evaluated spectrophotometrically at 544 nm after extraction, providing an effective means for quantifying these bioactive compounds⁴⁴.

Sugar metabolism

The sugars contents such as glucose, sucrose, and fructose in the grain were extracted using TAE buffer (50 mM; pH 7.5), with additives including polyclar (0.15%), sodium azide (0.02%), PMSF (2 mM), sodium bisulfite (NaHSO₃) (12 mM), mannitol (10 mM), and mercaptoethanol (1 mM), followed by centrifugation. The quantification of sugars was conducted using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), according to the method of Verspreet et al.⁴⁵. Maltotriose, not naturally present in the samples, was used as the internal standard to ensure the quality of extraction and purification, following AbdElgawad et al.⁴⁶.

Starch content was determined, following the methodology detailed by Galtier et al.⁴⁷, by involving the hydrating and gelatinizing the starch solution with 90% dimethyl sulfoxide (v/v), precipitating and washing it with ethanol and subsequently treating it with a mixture of α-amylase and amyloglucosidase to extract the starch.

Abdel-Mawgoud et al.⁴⁸ detailed the measurement of sucrose phosphate synthase (SPS) activity using a HEPES buffer (pH 8) with fructose-6-phosphate and UDP-glucose, incubated at 37 °C for 20 min, and terminated with NaOH (30%). AbdElgawad et al.²⁹ detailed the procedure for measuring invertase activity in solutions containing 100 mM sucrose in Na-acetate buffer (pH 5.0) with 0.02% (w/v) Na-azide.

Assessment of fatty acid, amino acid, and organic acid profiles in seeds

The fatty acid contents were measured in the grain based on the method of Torras-Claveria et al.⁴⁹. Briefly, samples were treated with methanol at room temperature, using nonadecanoic acid as the internal standard, and identified using the NIST 05 and Golm Metabolome databases.

The content of amino acids in the grain samples was determined based on the method of Zinta et al.⁵⁰. The samples were then homogenized in ethanol (80% v/v) and centrifuged at 14,000×g for 20 min. The supernatant was vacuum-evaporated, and the pellet was re-suspended in chloroform. Samples were then centrifuged for 10 min at 14,000×g. After that, the supernatant was filtered through a Millipore microfilter (0.2 µM pore size) and diluted with deuterium L-glutamine-2,3,3,4,4-d5 as the internal standard⁵⁰.

Organic acids were measured in the grain samples according to the method of AbdElgawad et al.²⁹. The supernatants were analyzed by high-performance liquid chromatography (HPLC) using 0.001 N sulfuric acid, set at 210 nm and a flow rate of 0.6 ml min^− 1, with an Aminex HPH-87 H column and a Bio-Red IG Cation H pre-column at 65 °C.

Polyamines metabolism

Total polyamines (PAs) contents were quantified using HPLC with a reversed-phase µ-Bondapack C18 column (Waters, USA), following the extraction of the samples with perchloric acid and subsequent hydrolysis with hydrochloric acid, as outlined by Shabbaj et al.⁵¹. For this process, approximately 1 gram of the plant tissue samples was homogenized in 5 mL of 0.4 M perchloric acid and centrifuged to obtain the supernatant, which was then neutralized with KOH and filtered for HPLC analysis. The PAs were separated on the HPLC system using a mobile phase optimized for polyamine detection, and their concentrations were determined based on peak areas. The enzyme activities associated with PA metabolism, including spermidine synthase (SPDS), arginine decarboxylase (ADC), and ornithine decarboxylase (ODC), were measured following the protocols established by Birecka et al.⁵² and Yoon et al.⁵³. The activities of ADC and ODC were determined through a radiolabeled carbon dioxide labeling technique, where the samples were incubated with specific substrates and the generated 14CO₂ was measured, yielding results expressed as nmol 14CO₂ mg^− 1 protein h^− 1. Moreover, the activity of SPDS was quantified using a fluorescence detection method via HPLC, with results reported as nmol mg^− 1 protein h^− 1.

Overall antioxidant measurement

Total antioxidant capacity (TCA) in shoot samples was determined using a spectrophotometric assay at 600 nm, where the plant materials were first homogenized in a suitable solvent and centrifuged to obtain a clear supernatant for analysis⁵⁴. For the quantification of reduced glutathione (GSH) and ascorbate (ASC) levels, HPLC was employed; samples were prepared by extracting the shoot tissues in 5% metaphosphoric acid, followed by centrifugation and filtration to yield a clear solution for HPLC analysis, utilizing appropriate mobile phases and conditions as detailed in the literature^55,56. Antioxidant enzyme activities, including peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR), were measured using various spectrophotometric methods that involved the integration of specific substrates and monitoring the change in absorbance over time at designated wavelengths^57–59. Furthermore, Glutaredoxin (Grx) activity was assessed through the reduction of 2-hydroxyethyl-disulfide, with the change in optical density measured spectrophotometrically. Thioredoxin (Trx) content was evaluated by monitoring NADPH oxidation, utilizing a spectrophotometric method that allowed for quantitative assessment of the thioredoxin levels in the samples^16,17.

Assessment of the content of phenolics and flavonoids

Quantifying individual flavonoids and phenolic acids was done using HPLC based on the method of Hegab et al.⁶⁰. To prepare the samples, approximately 1 gram of dried and powdered plant material was mixed with a 70% acetone-water solvent solution, which effectively extracted the polyphenolic compounds during a 30-minute sonication process at room temperature. The resulting mixture was then centrifuged at 4,000 rpm for 10 min, and the supernatant was carefully collected and filtered using a 0.45 μm nylon membrane filter to remove any particulate matter before HPLC analysis. The HPLC system was set up using a C18 reversed-phase column, and the individual flavonoids and phenolic acids were eluted using a mobile phase consisting of water containing formic acid (0.1%) and acetonitrile, under optimized gradient conditions that enabled the effective separation of the target compounds. The detection of components was conducted with a UV-Vis detector set at specific wavelengths corresponding to the maximum absorbance of each compound, which permitted precise quantification. Calibration curves were established before analysis using standard solutions of known concentrations of individual flavonoids and phenolic acids, allowing for accurate determination of the concentrations in the experimental samples based on peak areas acquired during HPLC runs.

Statistical analysis

All statistical analyses, including two-way ANOVA, Tukey’s HSD test, Ward’s clustering algorithm analysis, and graph generation, were conducted using R (ver. 3.5.1) and NCSS program (Version 21.0.3. Kaysville, Utah, USA). Results are presented as the mean of three replicates (n = 3).

Conclusions

The findings of the present research underscore the significant effects of OMWW treatment on wheat plants, especially under drought stress. The study showed that OMWW is a beneficial soil amendment that improves plant resilience and nutrition and can positively influence grain yield, nutritional quality, and functional properties. Furthermore, the increase in amylase activity and favorable modifications in lipid content and fatty acids profile suggest enhanced energy mobilization strategies essential for maintaining cellular integrity under stress conditions. The observed improvements in antioxidant enzyme activity, polyamine synthesis, and amino acid accumulation further highlight OMWW’s role in mitigating oxidative damage and stabilizing metabolic processes under drought stress. These findings provide mechanistic insights into how OMWW supports plant stress tolerance. Collectively, the findings suggest promising implications for sustainable agricultural practices, particularly in mitigating drought stress on wheat production. However, it is essential to critically assess the long-term sustainability of using OMWW as a soil amendment. Concerns regarding the potential accumulation of pollutants and their impacts on soil health must be addressed. Continued monitoring of soil and water quality and the long-term effects on crop productivity will be crucial to ensure that this practice remains effective and does not lead to adverse consequences over time. Therefore, while OMWW presents a viable approach to improve crop productivity, its implementation should be accompanied by rigorous assessments to maintain sustainability.

Author contributions

YAH and HS; Investigation, PS; Methodology, AMA and EAE; Resources, AMA, MOand EAE; Validation, MO and MSS Writing – original draft, YAH, MSS and HS; Writing – review & editing, YAH, HS, MHK, IAA, AE, MA, and PS . All authors reviewed the manuscript.

Funding

The work in this paper was supported, in part, by the Open Access Program from the American University of Sharjah. This paper represents the opinions of the author(s) and does not mean to represent the position or opinions of the American University of Sharjah. This work was funded by the Researchers Supporting Project number (RSP2025R176) King Saud University, Riyadh, Saud Arabia.

Data availability

The datasets used and/or analysed during the current study available from the corresponding authors on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.