Seed priming and irrigating with plasma-activated water improve the growth and drought resistance in Poa pratensis

Abstract

Background

Poa pratensis L. is a perennial grass commonly used for ecological restoration due to its rapid growth rate and strong adaptability. It is considered an excellent choice for soccer fields and urban green spaces because of its high wear resistance and durability. this is the first report demonstrating the co-application of seed priming and plasma-activated water irrigation for enhancing drought resistance in P. pratensis. This study aimed to examine the influence of seed priming and irrigation with PAW on the growth of P. pratensis and its ability to endure drought conditions. The experiment was conducted in a greenhouse in 2023, using a completely randomized design. Seeds were primed with either normal water (control group) or PAW and then sown in pots containing standard soil. The experiment included six treatments: three PAW management strategies—watered normally (WW, control group), seeds primed with PAW and irrigated with normal water (PW), and a combination of both methods (PP: primed and irrigated with PAW)—under both drought and non-drought stress conditions.

Results

The findings indicated that drought stress significantly decreased various growth parameters, including fresh weight (21% reduction), dry weight (27% reduction), chlorophyll levels (12% reduction), and the activities of the enzymes ascorbate peroxidase (APX) and catalase (CAT) (22% and 5% reductions, respectively) in P. pratensis. Conversely, drought stress increased the levels of several compounds: carotenoids (24% increase), malondialdehyde (MDA) (81% increase), proline (80% increase), soluble carbohydrates (15% increase), and the enzyme activity of guaiacol peroxidase (GPX) (36% increase). Under drought conditions, seed priming with PAW led to a decrease in MDA (21%) and an increase in fresh weight (approximately 13%) and dry weight (about 25%). Total chlorophyll increased by around 30%, while proline and soluble sugar content rose by 14% and 50%, respectively. The activities of APX, CAT, and GPX enzymes increased by 18%, 4%, and 11%, respectively. In summary, the combination of seed priming and irrigation with PAW under drought conditions reduced MDA content by 28% and enhanced plant biomass (fresh weight by 13% and dry weight by 21%), photosynthetic pigments (total chlorophyll by 17%), and osmoprotectants (proline by 56% and soluble carbohydrates by 11%). The activities of APX, CAT, and GPX also increased significantly—by 16%, 2%, and 16%, respectively.

Conclusions

PAW has been shown to improve drought stress in P. pratensis by reducing lipid peroxidation and increasing levels of photosynthetic pigments, osmoprotectants, and antioxidant enzyme activity. The results indicate that the most beneficial outcomes occur when seed priming is combined with PAW irrigation. This technology could serve as a cost-effective and sustainable method for enhancing growth and drought tolerance in plants, including P. pratensis, under water-stressed conditions. Further studies are necessary to explore this effect on additional plants and to better understand its details and possible mechanisms in future research.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07742-w.

Introduction

Poa pratensis L. (Poaceae) is a perennial grass known for its high quality, although it has poor drought resistance. It is found in diverse environments worldwide and is primarily used for forage and as turfgrass [1, 2]. This species exhibits better grazing tolerance compared to other cool-season forage grasses, making it an ideal choice for permanent pastures. P. pratensis has several beneficial characteristics, including a high growth rate, strong adaptability, and excellent resistance to wind [3]. Numerous studies have shown that P. pratensis can quickly cover soil surfaces, prevent soil erosion, improve soil structure, enhance soil carbon sequestration, and retain water and nutrients [4]. These attributes make it an excellent candidate for ecological restoration efforts worldwide. Currently, P. pratensis is widely utilized for restoring severely degraded grasslands, reclaiming mined areas, and supplementary sowing in lightly degraded grasslands, particularly in plateau regions [5].

Water stress affects approximately one-third of global agricultural land, significantly impacting crop performance [6]. The anticipated increase in global temperatures by 2 to 5 ^◦C by 2050, driven by climate change, is expected to exacerbate these issues, particularly in tropical, semi-arid, and coastal regions, thus threatening global food security [7]. This situation has made drought stress a critical global challenge, disrupting plant growth by altering biochemical and metabolic processes, resulting in substantial reductions in agricultural productivity [8, 9]. Drought negatively impacts all aspects of plant physiology by disrupting biochemical and physiological processes, leading to excessive production of reactive oxygen species (ROS) and triggering oxidative stress [10]. This oxidative stress causes photoinhibition, degradation of biomacromolecules, lipid peroxidation of membranes, and damage to DNA and proteins [11]. Turfgrass is an essential biotic component of urban and semi-urban ecosystems, playing a significant role in global carbon cycle research. During the dry season, drought and water scarcity can lead to a decline in turf quality, causing some leaves to wilt and reducing the ecological benefits provided by turfgrass. As a result, drought has become one of the primary environmental factors that limit the normal growth and development of turfgrass, negatively impacting its overall quality [12, 13]. Seed germination is a crucial stage in the plant growth cycle. Drought stress can limit water absorption and reduce the breakdown of seed storage compounds, which ultimately leads to a decrease in seed germination [14]. This stage is particularly critical in grasses because early growth stages are necessary for subsequent establishment. If, for any reason, the germination and initial growth of the grass are reduced, the weeds will have a chance to grow, which can significantly impact the grass’s quality [15]. There are several practical methods for seed priming, including hormonal priming, chemical priming, biopriming, osmo-priming, halopriming, and hydropriming. Many researchers have also studied physical priming to increase germination efficiency and improve adaptation to abiotic stresses [16–18].

Seed priming is a pre-sowing treatment often applied to commercial seed lots and is widely used by seed technologists to enhance seed vigour and seedling performance [19]. It is a controlled hydration process in which seeds absorb enough water to initiate early metabolic processes necessary for germination, without the formation of radicles [20]. This technique has gained significant attention due to its dual role in improving the early stages of plant growth and development, as well as providing a cost-effective strategy to mitigate the effects of biotic and abiotic stresses [21]. Seed priming is particularly beneficial under harsh environmental conditions, as it significantly enhances seed quality, which is a critical factor for successful crop establishment [22]. By activating “pre-germinative metabolism,” a set of metabolic pathways typically triggered during the early stages of germination, priming accelerates seedling emergence, improves germination rates, enhances seed resistance to various abiotic stresses, and increases the plant yield [23]. In this context, seed priming with plasma-activated water (PAW) can have significant effects on the quantity and quality of agricultural products, such as P. pratensis, under water stress conditions.

Conventional agricultural practices heavily depend on chemical fertilizers to enhance crop yields. However, this reliance has resulted in soil degradation and groundwater contamination [24, 25]. The overuse of these agrochemicals not only reduces nutrient use efficiency but also contributes to environmental issues such as eutrophication and greenhouse gas emissions [26]. To address these challenges, it is essential to optimize fertilization practices and explore alternative solutions, such as biofertilizers, nanotechnology [8, 10, 21], and plasma-based technologies [27–29]. PAW influences hormonal activity in plants, enhances water uptake, and modifies seed coats, resulting in improved sprout quality. These technologies affect bioactive compounds such as proteins, carbohydrates, enzymes, polyphenols, gamma-aminobutyric acid, and antioxidants, which support seed growth and change the nutritional and functional properties of sprouts. PAW, due to its unique chemical characteristics, acidifies the environment, modifies redox potential, and generates reactive oxygen and nitrogen species, all of which are crucial for metabolic pathways involved in seed germination [30].

A plenty of researchers have investigated [31] the applications of plasma technology in agriculture and found that the seed germination rate [28, 32–38], plant growth rate [28, 32–35, 37, 39–43] and crop yield [34, 37, 42, 43].

Drought stress is one of the most significant abiotic stresses that affects grass growth worldwide, and the reduction in grass quality due to drought stress is a major concern for grass management. However, the growth of P. pratensis is adversely influenced by shortage of irrigation and drought, as well as high temperatures induced by global climate change [44]. One of the methods used to increase the resistance of plants to biotic and abiotic stresses is the use of growth regulators. However, some of these regulators are expensive, not cost-effective, and have harmful long-term effects. Currently, interest in exploring low-cost and eco-friendly methods is increasing. However, there have been no studies on the use of cold plasma techniques to increase growth and stress resistance in grass. Therefore, the objective of this research was to investigate the impact of PAW on the growth and drought resistance of Super Sport, a cool-season grass species.

Despite extensive research on the role of (PAW) in mitigating environmental stresses, its application in seed priming and irrigation has not been fully explored, particularly in plants like P. pratensis. This grass species is widely used for ecological restoration due to its high growth rate and strong adaptability. This knowledge gap underscores the need for further studies to assess the potential of PAW in enhancing plant performance under drought conditions. Therefore, this study aims to evaluate the effects of seed priming and irrigation with PAW on the growth, biochemical, and physiological traits of P. pratensis when subjected to water stress. The primary focus is on plant growth, antioxidant activity, and the reduction of oxidative stress. This research is pioneering in its exploration of PAW’s effects on P. pratensis under drought stress, providing valuable insights into using PAW to improve plant resilience and productivity in challenging environments. The findings could significantly advance sustainable agriculture and enhance stress resistance, as the use of PAW in seed priming presents a promising approach to reduce reliance on chemical fertilizers and boost agricultural productivity.

Materials and methods

This research complied with relevant institutional, national, and international guidelines and legislation of Iran. P. pratensis seeds were provided by the Pakan Bazr Company (Isfahan, Iran). The soil used in this experiment was collected from a location in Kerman, Iran, characterized by Clay sandy texture, and the physical and chemical characteristics presented in Table 1.

Table 1.

Physicochemical characteristics of the soil before implementing treatments

Soil Texture	pH	EC (ds/m)	Organic Carbon (%)	K (mg.kg-1)	P(mg.kg-1)	N (%)
Clay Sandy	7.44	1.90	1	320	14.8	9

Plant materials and treatment

Fifteen grams of P. pratensis seeds of uniform size were soaked in 200 ml of either normal water (control group) or PAW (which has been treated with plasma for 20 min) for 20 h at room temperature in the dark with continuous aeration for each treatment. This time was optimized in a preliminary experiment. The primed seeds were planted in pots filled with a clay‒sand mixture. The plants grew in a greenhouse at 16/8 h light/dark period, thermoperiod of 25/22°C day/night, 70% relative humidity, and the photon flux density was 200 µmol m⁻² s⁻¹. While CO₂ levels were not specifically monitored, the consistent use of natural ventilation through roof vents would have maintained ambient atmospheric CO₂ concentrations, minimizing its potential as a confounding variable. Before germination, the pots were sprayed daily with either regular water or PAW (post-activated water). After the seeds germinated, the plants received 200 ml of either PAW or regular water every other day for two weeks. To induce drought stress, watering was stopped for three days. After this period of drought stress, the plants, now three weeks old, were harvested to measure growth and physiological parameters. In this experiment, there were three primary treatment groups. The first group was the control group of plants (WW). In the second group, the seeds were primed with PAW and irrigated with regular water (PW). Finally, in the third group, the plant seeds were primed with PAW and irrigated with PAW (PP). Each treatment group was further divided into subgroups. These subgroups were subjected to either drought stress (D), where they were not watered for three periods, or normal conditions (C), where they were watered every other day. These treatments are summarized in Table 2. Overall, the experiment was arranged in a completely randomized design with 6 treatments and 4 biological replicates per treatment. Each replicate consisted of a single pot sown with 30 seeds. In total 24 experimental units were used. These treatments are summarized in Table 2.

Table 2.

A summary table of experiments treatments

Drought	treatment
-	WW(control group): priming and irrigation with tap water
-	PW: priming in PAW and irrigation with tap water
-	PP: priming in PAW and irrigation with PAW
+	WW (control group): priming and irrigation with tap water
+	PW: priming in PAW and irrigation with tap water
+	PP: priming in PAW and irrigation with PAW

To induce drought stress, the plants were not irrigated for three days before harvesting the samples

Plasma activated water preparation

Our homemade plasma reactor generated PAW. The reactor consisted of two coaxial cylinder electrodes, each 15 cm long. The inner electrode, made of stainless steel, had a diameter of 10 mm. The outer electrode was fashioned from copper foil wrapped around a Pyrex tube. A pulsed DC high voltage with a frequency of 200 Hz and adjustable pulse height biased the electrodes. A 2-millimeter-thick Pyrex tube served as a dielectric, preventing current spikes. This configuration produced a dielectric barrier discharge (DBD) within the air-filled gap between the electrodes. A digital flow controller (Breezens GP Series) regulated the air flow rate. To dissolve the resultant gases in water, a diffuser at the bottom of a cylindrical water container received the treated air (Fig. 1a and b). We prepared the PAW samples by treating 1.0 L of distilled water in the container for 20 min. To determine the concentration of oxygen and nitrogen species, a setup consisting of a spectrometer (AVANTES Ava Spec), homemade absorption cell, cuvette holder, and a UV light source (Bloor Azma Deuterium Light Source) was utilized (Fig. 1. c and d). The concentration of reactive species were calculated with standard curve obtained by Noori et al. 2021 [45].

Fig. 1.

a: Schematic image of a homemade plasma reactor was used to prepare ozone gas, b: A homemade plasma reactor which was made by two coaxial cylinder electrodes with the length of 15 cm, c: Schematic image of a homemade absorption cell, and a UV light source, d: A homemade absorption cell, and a UV light source (BloorAzma Deuterium Light Source) was used in this experiment

Fresh and dry weight of shoot

To measure plant weight, 10 plants were chosen from each pot as replicates, and the shoots of the plants were weighed. The samples were then dried in an oven at 50 °C for 24 h, after which the dry weights were measured.

Photosynthetic pigments of leaves

The measurement of photosynthetic pigments, including chlorophyll a, b and total chlorophyll and carotenoids (carotenoid and xanthophyll), was performed according to the methods of [46]. The process involved homogenizing 0.2 g of plant sample in 15 milliliters of 80% acetone. The resulting supernatant was then measured for its absorbance at 646.8 nm, 663.2 nm and 470 nm to determine the levels of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids.

 

 

 

Lipid peroxidation

To calculate the malondialdehyde (MDA) content, the value of nonspecific absorption at 600 nm was subtracted from the 532 nm reading [47]. The MDA content was expressed as µmol MDA per g fresh weight and calculated with an extinction coefficient of 155 mM⁻¹ cm⁻¹.

Soluble carbohydrate

To determine the total amount of soluble sugar, anthrone reagent was used with glucose as the standard. First, 6 mL of anthrone reagent (150 mg of anthrone in 72% H₂SO₄) was added to the leaf tissue extract. The mixture was then heated in a water bath at 100 °C for 10 min. After that, the test tubes were cooled on ice for 10 min and incubated for 20 min at 25 °C. The absorbance was then read at 625 nm. To create the standard curve, various glucose concentrations were used [48].

Proline: First, 0.2 g of frozen plant tissue was homogenized in 10 mL of 3% sulfosalicylic acid and centrifuged. the supernatant was mixed with ninhydrin reagent and toluene. The proline content was determined at 520 nm and a standard curve was used to calculate the amount of proline [49].

Enzyme extraction and activity determination

After 300 milligrams of shoot fresh weight were homogenized with 3 mL of 50 mM potassium phosphate buffer, the suspension was centrifuged at 10,000 × g at 4 °C for 20 min, and the supernatants were used for enzyme activity measurements by considering protein amounts. The Bradford method was used for determination of the protein amounts in the plant samples [50].

Catalase Activity (CAT) (EC 1.11.1.6)

CAT (EC 1.11.1.6) activity was measured via the method of [51]. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 15 mM H₂O₂, and 100 µL of the enzyme extract. The decrease in the absorbance of the mixture was determined at 240 nm (Ɛ=40 mM^{− 1} cm^{− 1}). The enzyme activity was expressed in U per milligram of protein (one unit = 1 micromole of H₂O₂ reduction per min per mg protein).

Guaiacol Peroxidase (GPX) (EC1.11.1.7)

The reaction mixture consisted of 50 mM potassium phosphate (pH 7.0), 0.3% (v/v) H₂O₂, 1% (v/v) guaiacol, and 100 µL of enzyme extract. The enzyme activity was recorded as U per milligram of protein, where one unit (U) of enzyme activity was considered the amount of enzyme that produced 1 micromole of tetra guaiacol per minute [52].

Ascorbate Peroxidase (APX) (EC 1.11.1.11)

A mixture of 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, and 0.1 mM H₂O₂ was prepared in a test tube. Then, 150 µl of enzyme extract was added to the mixture, and the absorbance was recorded at 290 nm with Ɛ=2.8 mM^{− 1} cm^{− 1}. The enzyme activity was expressed in U per milligram of protein [53].

Statistical analysis

The experiment was conducted in a factorial arrangement (two factors: plasma and water stress) based on a completely randomized design (CRD) with four replications. Analysis of variance (ANOVA) and mean comparisons using the LSD test (P ≤ 0.05) were performed with SAS 9.4 software. Prior to ANOVA, data were tested for normality using SAS and for homogeneity of variances using Levene’s test in SPSS 22.0 to ensure the assumptions of the analysis were met.

Results

Plasma activated water analysis

The analysis results of plasma-activated water are presented in Table 3. The data indicate the presence of active oxygen and nitrogen species in the water; however, their concentrations differ.

Table 3.

Compounds produced in plasma activated water

species generated	20 min
O2	8 ppm
O3	5.1 ppm
H2O2	6.4 ppm
NO-3	1.87 ppm
NO-2	0.15 ppm

The ANOVA results are presented in Tables 4 and 5. Based on this analysis, water stress and plasma treatment significantly affected all measured traits. The interaction between plasma and water stress was significant only for carotenoids, sugar, and proline. This indicates that while most traits respond independently to each factor, the combined effect of plasma and water stress specifically influences the accumulation of certain biochemical compounds, suggesting a potential synergistic response in these traits.

Table 4.

Analysis of variance (ANOVA) for growth parameters and photosynthetic pigments of P. pratensis under PAW and drought treatments

Source	DF	fresh weight	dry weight	chlorophyll a	chlorophyll b	total chlorophyll	carotenoids
drought	1	0.667**	0.015 **	0.432**	0.042*	0.459**	38111.743**
PAW	2	0.078**	0.002**	0.610**	0.035*	0.631**	8657.831**
drought × PAW	2	0.014 ns	0.000 ns	0.010 ns	0.002 ns	0.010 ns	1131.562**
Error	18	0.008	0.000	0.021	0.008	0.021	102.5
CV	-	5.36	5.18	6.16	14.61	6.03	4.8

PAW plasma-activated water, DF degrees of freedom, CV coefficient of variation

ns non-significant; *: significant at p ≤ 0.05; **: significant at p ≤ 0.01

Table 5.

Analysis of variance (ANOVA) for some physiological parameters of P. pratensis under PAW and drought treatments

Source	DF	MDA	soluble sugars	proline	GPX	APX	CAT
drought	1	0.109**	198.03**	54.15**	1337.88**	27.93**	22.72**
PAW	2	0.013**	353.5**	5.54**	118.50**	4.07 **	2.96**
drought × PAW	2	0.001 ns	171.26**	1.88**	1.39 ns	0.85 ns	0.22 ns
Error	18	0.0003	2.403	0.036	7.67	0.883	0.144
CV	-	7.99	4.31	4.45	5.53	7.56	0.85

PAW plasma-activated water, DF degrees of freedom, CV coefficient of variation

ns non-significant; *: significant at p ≤ 0.05; **: significant at p ≤ 0.01

Biomass parameters

Fresh and dry weight of shoot: The results show that, compared to the control treatment, drought stress reduced the fresh weight of the plants by approximately 21% and the dry weight by 27%. Under non-drought conditions, the application of PAW priming increased fresh weight by 4% and dry weight by 5%. Additionally, the combination of seed priming and irrigation with PAW resulted in significant increases in fresh weight (14%) and dry weight (11%) under non-drought conditions. Under drought stress, seed priming with PAW increased fresh weight by 12.6% and dry weight by 25%. The combination of seed priming and PAW irrigation also enhanced plant biomass under drought conditions, leading to an increase in fresh weight of 13.3% and dry weight of 21% (Fig. 2B). Comparison of the two PAW application methods under none-stressed conditions showed that co-application of seed priming and irrigation methods with PAW had a significant increase in fresh and dry weight. However, under drought stress, no significant difference was observed between the two PAW application methods in biomass Parameters.

Fig. 2.

Effect of PAW on the shoot fresh weight (A) and dry weight (B) of P. pratensis under drought stress. WW = control group; PW = seeds primed with PAW and irrigated with normal water; PP = seeds primed with PAW and irrigated with PAW. The data are presented as the means ± SE of four replicates. Columns with different letters indicate significant differences according to LSD test at p ≤ 0.05

Photosynthetic pigments (chlorophyll a, b, total chlorophyll and carotenoids)

Drought stress led to a significant decrease in chlorophyll content, specifically chlorophyll a (12.3%), chlorophyll b (9%), and total chlorophyll (12%), compared to conditions without drought stress. The application of PAW priming treatment resulted in an increase of 23% in chlorophyll a, 21% in chlorophyll b, and 23% in total chlorophyll under non-drought stress conditions. When combining seed priming with irrigation using PAW, there were notable increases in chlorophyll a (18%), chlorophyll b (26%), and total chlorophyll (18%) under non-drought stress conditions. In drought stress conditions, seed priming with PAW increased chlorophyll a by 30%, chlorophyll b by 9.4%, and total chlorophyll by 29%. Additionally, under drought stress, the combination of seed priming and PAW irrigation resulted in increases in chlorophyll a (17%), chlorophyll b (11%), and total chlorophyll (17%) (see Fig. 3A-C). Drought stress caused an increase in carotenoids (24%). Application of PAW priming treatment and the combined effect of seed priming and irrigation with PAW resulted in a significant reduction (29% and 39% respectively) in carotenoids content under non-drought stress conditions. Also, under drought stress, PAW pretreatment and the combination of seed priming and irrigation with PAW reduced carotenoids content (12% and 13% respectively) (Fig. 3D). Comparison of the two PAW application methods under none-stressed conditions showed that co-application of seed priming and irrigation methods with PAW did not have a significant difference in chlorophyll content. However, under drought stress, the PAW priming application method had a significant increasing effect on photosynthetic pigments.

Fig. 3.

Effects of PAW on the chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) and carotenoid (D) contents of P. pratensis under drought stress. WW = control group; PW = seeds primed with PAW and irrigated with normal water; PP = seeds primed with PAW and irrigated with PAW. The data are presented as the means ± SEs of four replicates. Columns with different letters indicate significant differences according to LSD test at p ≤ 0.05

Lipid peroxidation

MDA content

The measurement of malondialdehyde (MDA), an indicator of membrane lipid peroxidation, showed that drought stress significantly increased MDA levels by 81%. In plants exposed to drought stress, seed pretreatment with PAW reduced MDA levels by 21%. Furthermore, applying PAW irrigation after seed pretreatment led to a 28% decrease in MDA content (Fig. 4 A). Under non-drought stress conditions, treating seeds with PAW or a combination of PAW treatment and irrigation resulted in a significant reduction in MDA content. Comparison of the two PAW application methods under none-stressed and drought stress conditions showed that no significant difference was observed between the two PAW application methods in MDA content.

Fig. 4.

Effects of PAW on the MDA (A), proline (B) and soluble sugar (C) contents of P. pratensis under drought stress. WW = control group; PW = seeds primed with PAW and irrigated with normal water; PP = seeds primed with PAW and irrigated with PAW. The data are presented as the means ± SE of four replicates. Columns with different letters indicate significant differences according to LSD test at p ≤ 0.05

Osmolytes (proline and sugars)

Proline

The findings revealed that drought stress increased proline levels by approximately 80% compared to non-drought conditions. Under drought stress, the application of PAW priming treatment and the combination of seed pretreatment with PAW irrigation significantly increased proline content by 50% and 56%, respectively, compared to untreated plants (Fig. 4B). Even under non-drought conditions, the application of PAW priming treatment and the combined effect of seed priming with PAW irrigation led to significant increases in proline content of 16% and 33%, respectively. Comparison of the two PAW application methods under none-stressed conditions showed that co-application of seed priming and irrigation methods with PAW had a significant increase proline content. However, under drought stress, no significant difference was observed between the two PAW application methods in proline content.

Soluble carbohydrate content

The results indicated that the soluble carbohydrate content increased by 15% under drought stress. The application of PAW priming treatment, along with the combination of seed priming and irrigation with PAW, further enhanced the soluble carbohydrate levels by 14.3% and 11.3%, respectively, under drought stress (see Fig. 4 C). Additionally, treating seeds with PAW, as well as the combined approach of seed priming and irrigation with PAW, significantly increased the amounts of soluble sugars by 58% and 83%, respectively, under non-drought stress conditions. Overall, although under none-stress conditions, comparing the effect of co-application of priming and irrigation with PAW resulted in a significant increase in sugar content compared to priming, but under drought stress conditions, no significant difference was observed between the two PAW application methods.

Antioxidant enzyme activity

The results indicated that drought stress caused a decrease in the activity of catalase (CAT) by 5% and ascorbate peroxidase (APX) by 22%, while the activity of guaiacol peroxidase (GPX) increased by 36%. Seed priming with PAW resulted in increases in APX, CAT, and GPX activities by 18%, 4%, and 11%, respectively. Additionally, treating seeds with PAW, in conjunction with irrigating with PAW, led to increases in enzyme activities of APX (16%), CAT (2%), and GPX (16%) (Fig. 5 A and 5B, and 5 C). The application of the PAW priming treatment, as well as the combined use of seed priming and irrigation with PAW, enhanced enzyme activities even under non-drought stress conditions. Comparison of the two PAW application methods under none-stressed conditions, no significant difference was observed between the two PAW application methods in studied enzyme activities. However, under drought stress, except for the PAW priming application method, which had a significant increasing effect on catalase enzyme activity, no significant difference was observed between the two PAW application methods in other antioxidant enzyme activities.

Fig. 5.

Effect of PAW on the activity of the antioxidant enzymes CAT (A), APX (B) and GPX (C) in P. pratensis under drought stress. WW = control group; PW = seeds primed with PAW and irrigated with normal water; PP = seeds primed with PAW and irrigated with PAW. The data are presented as the means ± SE of four replicates. Columns with different letters indicate significant differences according to LSD test at p ≤ 0.05

Discussion

The findings of this study revealed a decrease in growth parameters and photosynthetic pigments, alongside an increase in lipid peroxidation under drought stress. This pattern was also observed in P. pratensis and Lolium perenne [54]. Chlorophylls play a crucial role in light absorption, energy transfer, and electron transport during photosynthesis. Under drought conditions, chlorophyll content typically declines due to the accumulation of reactive oxygen species (ROS) [55]. The presence of ROS induces lipid peroxidation in cell membranes, leading to chloroplast damage and an increase in chlorophyllase activity, which ultimately results in the degradation of chlorophyll precursors [10, 11]. Additionally, drought stress disrupts the transfer of essential nutrients for growth. This disruption results in reduced dry weight and overall growth, as the production of new dry matter decreases [56]. Our findings indicate that seed priming and the combination of priming with irrigation using PAW can alleviate the negative effects of water stress on chlorophyll content. This approach enhances plant biomass (both fresh and dry weight), improves the levels of photosynthetic pigments and osmoprotectants (such as proline and soluble carbohydrates), increases the activity of antioxidant enzymes, and reduces the lipid peroxidation index, leading to improved plant growth and drought resistance in P. pratensis under both non-stressed and drought-stressed conditions. Similar results have been reported in Lactuca sativa [57], Fagopyrum esculentum, and Hordeum vulgare [32].

PAW is produced through the reaction of nonthermal plasma with water, leading to the generation of active oxygen and nitrogen species. This environmentally friendly method offers a viable alternative to traditional techniques [58]. Research indicates that the effects of PAW on plant growth relate closely to its chemical composition, particularly its active oxygen and nitrogen species. These reactive species generated in PAW can act as positive signaling molecules, helping to reduce seed dormancy and promoting seed germination and overall plant growth [59]. Moreover, PAW—often referred to as “plasma fertilizer”—is known to contain high levels of nitrates and nitrites, which are essential nutrients for plant development [28, 41, 60]. Studies have shown that PAW treatments can increase the content of photosynthetic pigments (chlorophyll a and b) and enhance the photosynthetic rates in lettuce [40]. In addition, the use of PAW as a priming treatment has led to a significant increase in chlorophyll concentration in various plants [34, 39, 42, 61]. In present research, there has also been a notable rise in carotenoid content in P. pratensis plants subjected to drought stress. Carotenoids play a vital role in photosynthesis and in protecting plants from light damage. The observed increase in carotenoids during severe drought conditions helps safeguard against chlorophyll degradation caused by light oxidation. It seems that the upregulation of the carotenoid biosynthetic pathway as a protective mechanism against reactive oxygen species (ROS) and to dissipate excess light energy. Similar enhancements in carotenoid content under drought stress have also been reported in chamomile [62]. Although the pretreatment with PAW did not significantly alter the amounts of carotenoids produced in this particular study, the foliar application of PAW in field experiments with rice was found to significantly boost the total contents of chlorophyll and carotenoids. Content [34].

The level of malondialdehyde (MDA) resulting from the lipid peroxidation of membrane lipids is commonly used as an indicator of oxidative damage [63]. The results of the present study indicated an increase in MDA content under water stress, which is consistent with previous research showing that elevated drought stress leads to higher MDA levels in African grass (Cynodon dactylon) [64]. Furthermore, our study found that treating P. pratensis plants with PAW resulted in a reduction in MDA content. Notably, a significant decrease in MDA levels in mung bean plants following PAW treatment has also been reported [65] These researchers suggested that PAW treatment can mitigate lipid peroxidation damage by enhancing the activity of antioxidant enzymes [65]. This hypothesis is further supported by the increase in antioxidant enzyme activity observed in the P. pratensis plants treated with PAW in this study. Additionally, research by Adhikari et al. (2019) on tomato plants showed that those irrigated with PAW for 15 and 30 min exhibited less damage compared to the control group [66].

In this study, we observed a significant increase in proline content in plants subjected to drought stress. The rise in proline concentration is a form of adaptation that helps plants cope with stressful conditions [67, 68]. Under stress, proline performs various vital functions, such as maintaining osmotic balance, protecting the structure of proteins and cell membranes, stabilizing intracellular components, and neutralizing free radicals. Furthermore, proline acts as a reservoir for carbon and nitrogen [69]. Research on drought tolerance in various grass species, including Agropyron desertorum and Festuca arundinacea, has demonstrated that drought stress leads to increased proline levels in these plants [70]. The findings of this study indicated that pre-treating P. pratensis with PAW resulted in elevated proline content. One possible explanation for the higher proline levels in plants pre-treated with PAW is improved access to nitrogen sources.

Soluble sugars act as osmolytes that accumulate in plants in response to drought and salinity stress, helping to regulate osmosis. Research has indicated that sugars play a vital role in maintaining the structure of macromolecules within cells and stabilizing DNA. Some scientists have pointed out that the levels of soluble sugars can serve as a reliable indicator of a plant’s resistance to drought and salinity stress [71]. Additionally, total soluble sugars offer an adaptive response to various other stresses, including pathogen attacks, low temperatures, and anoxic injury [72]. In the present research, an increase in soluble sugar levels has been observed in P. pratensis plants following plasma treatment. Similar findings were reported in pea plants by Rathore et al. (2022). The effects of PAW treatment on enhancing soluble sugars in other plants have also been documented [34, 72].

The production of reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), increases in plant cells during biotic and abiotic stress. High concentrations of ROS can lead to oxidative damage within cells [35]. Antioxidant enzymes play a crucial role in helping plants respond to environmental stresses [73]. In this study, the activities of catalase (CAT) and ascorbate peroxidase (APX) enzymes decreased in P. pratensis plants under drought conditions. Similar reductions in CAT and APX activities have also been reported in tall fescue (Festuca arundinacea L.) and Kentucky bluegrass (P. pratensis L.) under drought stress [74]. CAT and APX are important for detoxifying hydrogen peroxide through their antioxidant functions and for blocking the chain reactions involving free radicals [75]. A decrease in the activities of CAT and APX can lead to an accumulation of hydrogen peroxide, which can carry out the Haber-Weiss reaction and lower the activity of certain enzymes in the Calvin cycle, such as ribulose monophosphate kinase and phosphoribulo kinase. A decline in the activity of these enzymes can further increase the production of reactive oxygen species and damage biomolecules, while also reducing the NADP+/NADPH + ratio and H + concentration in the chloroplasts [76]. The results of this study indicated an increase in the activity of the glutathione peroxidase (GPX) enzyme in P. pratensis plants subjected to drought stress. The GPX enzyme helps remove reactive oxygen species during periods of drought. Guaiacol serves as an electron donor, using hydrogen peroxide to oxidize various organic and inorganic substrates. The activity of GPX, along with other cytosolic defense mechanisms, significantly enhances the plant’s resilience during stress. An increase in GPX activity has also been observed in perennial ryegrass (Lolium perenne) [77].

In this study, a decrease in catalase (CAT) and ascorbate peroxidase (APX) activity was observed alongside an increase in guaiacol peroxidase (GPX) activity. Importantly, not all antioxidant enzymes show an increase during drought stress. The response varies based on factors such as the type and concentration of stress, the plant species, and the growth stage of the plant [78]. The PAW treatment in this study notably enhanced the activity of antioxidant enzymes, including CAT, APX, and GPX. This observation aligns with previous findings in maize, tomato, pepper [28], and lettuce [35]. The active species present in PAW probably promote the accumulation of secondary metabolites with antioxidant properties, such as phenolics and carotenoids, as well as the expression and activity of antioxidant enzymes (GPX, APX, and CAT) that help mitigate the effects of reactive oxygen species (ROS), thereby triggering a defensive response [79, 80]. The observed increase in CAT, ascorbate, and GPX enzyme activity may be attributed to the reduction of excess ROS through treatment with plasma-activated water, which contains a specific amount of ROS along with significantly higher concentrations of active nitrogen species [61, 68]. These findings may help explain the observed increase in growth and resistance to drought stress in P. pratensis.

Conclusions

In this study, the application of PAW has demonstrated significant potential in alleviating drought stress in P. pratensis plants. The protective effects of PAW may be linked to increases in various growth parameters, including fresh(13%) and dry weight (21%), as well as improvements in photosynthetic efficiency (17%), levels of osmoprotectants (such as soluble sugars (11%) and proline (56%)), and the activity of antioxidant enzymes (16%)(including catalase CAT, ascorbate peroxidase APX, and guaiacol peroxidase GPX(. Additionally, a significant decrease in malondialdehyde (MDA) content was observed. Therefore, utilizing PAW as a novel approach for seed priming or irrigation can help mitigate these abiotic stresses and lessen the negative effects of drought. The combination of seed priming and irrigation using PAW may yield the greatest benefits for plant growth and improvement under drought conditions. PAW is presented as an innovative, eco-friendly alternative for crop nutrition through irrigation, as it does not rely on chemical additives, enhances germination, and stimulates plant growth and defense mechanisms. However, this experiment was conducted under controlled greenhouse conditions, indicating the need for further research in field experiments. To thoroughly understand the mechanisms behind these positive interactions, more in-depth studies are necessary. From a practical perspective, conducting economic analyses to evaluate the cost-benefit ratio of this technology in real agricultural settings, as well as feasibility studies for its implementation and commercialization, will be crucial. Long-term assessments of its impact on agricultural sustainability are also essential for the development of innovative and sustainable solutions in modern agriculture.

Abbreviations

APX

Ascorbate peroxidase

Car

Carotenoid

CAT

Catalase

Chl

chlorophyll

DBD

Dielectric barrier discharge

GPX

Guaiacol peroxidase

MDA

Malondialdehyde

PAW

Plasma-activated water

ROS

Reactive oxygen species

Authors’ contributions

This research was performed in collaboration with all the authors. F. N supervised and planned the study and contributed to the interpretation of the data, writing, review, and editing of the manuscript. M. Z, F. R and E. AM carried out the experiments, material preparation, and data collection. F. NA, B. K, and E. AM analyzed the data and wrote the manuscript. All the authors reviewed and approved the final manuscript.

Funding

This research was financially supported by Shahid Bahonar University of Kerman, Kerman, Iran.

Data availability

The data that support the findings of this study are available from the first author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable. This manuscript does not involve research on humans or animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.