The Effect of Plasma-Activated Water on Zea mays L. Landraces Under Abiotic Stress

Abstract

A major challenge in the agricultural industry is finding innovative and sustainable methods that can lead to enhanced crop resistance to abiotic stress factors and increased productivity. Research in recent years has proven the potential of non-thermal plasma in various fields, including agriculture, with relevance in promoting plant growth and development, plant immune response to abiotic stress or pathogen resistance. In the present study, distilled water was activated using dielectric barrier discharge equipment; subsequently, plasma-activated water (PAW) was used to irrigate maize plants subjected to cold stress. Two different maize accessions were studied in this work, SVGB-11742 and SVGB-718, previously identified as highly and moderately resistant to cold stress, respectively. After plant exposure to cold and irrigation with plasma-activated water, morphological, morpho-agronomical and physiological parameters and molecular data were assessed. The two genotypes showed distinct, often opposing, responses to PAW treatment depending on the parameter assessed. Generally, the obtained data at the molecular level showed that treatment with PAW increased the expression of certain genes involved in growth and development of the SVGB-718 variant subjected to cold stress. Irrigation of plants exposed to low temperatures with PAW did not have the predicted effects at the morphological and even the physiological level regarding the concentration of assimilatory pigments and the cold test index. While morphological benefits were limited and genotype-specific, PAW induced significant molecular changes (upregulated stress-responsive genes in SVGB-718), suggesting a priming effect that may not have been captured in the short-term morphological assays. However, the results obtained represent an important background for future studies.

1. Introduction

Originating from tropical and subtropical regions, maize (Zea mays L.) is an annual grass, considered one of the most important cereal grains worldwide [1,2]; moreover, maize is a potential food security crop, with an important role in providing food and nutrition security for communities globally [3]. With a harvested area of approximately 209 million hectares worldwide and yield of about 5.9 thousand kg/ha in 2023, maize is a staple food grown in almost all countries of the world [4]. According to the National Institute of Statistics (NIS Romania), in 2018, Romania ranked first in Europe for maize production, while in 2022 it ranked second, following France. Maize seeds have an important nutritional profile, as they are rich in carbohydrates, representing an important source of energy for consumers, providing proteins, lipids and a variety of vitamins and minerals [5].

Along with gases, liquids and solids, plasma is the fourth state of matter. Plasma is characterized as a partially ionized gas consisting of ions, neutral particles and electrons [6]. Plasma is the most abundant state of matter in the universe, constituting over 99% of visible matter, excluding planets and other cold celestial bodies [7], but it can also be generated under laboratory conditions. There are several non-thermal plasma generation techniques, such as dielectric barrier discharges (DBDs), jets, torches, corona and spark discharges, direct current discharges or radio-frequency discharges [8]. Over the years, studies have shown that non-thermal plasma has paramount potential in various domains, such as agriculture [8,9], medicine [10,11] or the food industry [12,13]. The generation and usage of non-thermal plasma in agriculture is seen as an environmentally friendly technology, which makes it an excellent alternative for stimulating plant germination and growth [14,15,16], substituting chemical fertilizers that persist in the soil and contaminate the environment [8] and increasing the resistance of plant species to various biotic [17] and abiotic stress factors [18]. The vegetal material can be exposed directly to the plasma gas or indirectly, by treating the water, which is then used to irrigate the plants.

PAW’s effectiveness depends on whether it is used immediately after production or whether it is stored and used after some time. Several studies indicate a decrease in pH values after different periods of storage; for instance, after 70 h of storage, there is a decrease from 2.07 to 1.5 [19], or there is a decrease from 6.8 to 2.3 after 30 days of storage [20], regardless of the storage temperature. Shen et al. [20] analyzed the antimicrobial potential of PAW after storage in different conditions and concluded that a temperature of −80 °C best conserved this capacity, while temperatures of +25/+4/−20 °C showed a decrease in this activity. Several nitrogen and oxygen reactive species contribute to PAW’s antimicrobial activity by causing membrane and cellular wall damage and, they also affect DNA and protein integrity [21]. Some researchers recommend keeping PAW for an hour or even a day at room temperature before treating seeds to reduce short-life reactive oxygen and nitrogen species [22]. When using indirect treatment, immersing the seeds in PAW before sowing may affect the seed microbiome [23]. The microbiome plays a crucial role in seed physiology, as it influences several key aspects of plant development and health. The seed-associated microbiome can enhance germination by producing various phytohormones (auxins, gibberellins), improve the resilience of abiotic and biotic stress factors, contribute to the mobilization and uptake of nutrients (nitrogen, phosphorus) essential for early seedling growth and help in root and shoot development by interacting with plant signaling pathways [24,25,26]. The normal bacteria population can suffer various changes when exposed to plasma or plasma-activated water, and the effects produced by these kinds of treatments can also be correlated with alterations to the microbiome. For instance, Mravlje et al. [27] exposed common and Tartary buckwheat seeds to direct cold plasma treatment for different periods of time, from 15 to 120 s, in a low-pressure radio-frequency treatment and proved that the treatments which exceeded 60 s negatively affected the germination percentage, while a reduction in fungal population was registered after exposures of 90 and 120 s. The alteration in the seed microbiome caused by various plasma treatments is associated with consequences of germination and viability, and it is important to define optimum exposure parameters in order to ensure global food security.

Climate change, the demand for quality food and the necessity to provide an equal distribution of food worldwide place the agricultural sector in a critical situation. There is a real challenge and an urgent interest in identifying ecologically and financially profitable ways to develop plant species of interest, with increased resistance to biotic and abiotic stress. According to The United Nations, by 2050, the global population will reach 10 billion, and the agricultural community must come up with solutions to avoid food insecurity and provide access to safe and nutritious staple food [28].

A suboptimal temperature at any growth stage of a plant can negatively affect the plant’s development and thus its yield production [29]. When the maize crop is exposed to temperatures between 0–15 °C, cold stress can occur, which may produce serious damage to the plants [30]. It is difficult to sow maize in temperate regions. Generally maize is sown earlier to avoid exposure to drought or pests and to accelerate biomass accumulation. Still, this can subject maize to cold stress, a phenomenon that can have a negative impact, especially if it appears in the first stages of development [31]. Early sowing of maize involves several risks, including the probability of the crop being damaged more or less intensively by low overnight temperatures. Thus, the presence of cold stress can severely affect the maize crop by altering certain morpho-agronomic parameters, such as growth vigor, cold resistance, silking time, flowering time, breaking plants, plant fall, number of sterile plants, Fusarium resistance and weight of 1000 seeds. Obviously, all these changes have a high impact on the crop yield [32]. Numerous studies have shown that cold stress may damage seed germination by reducing the germination percentage and the growth rate of germinated seeds [33,34,35]. When temperatures are lower than optimal growing, several morphological changes can occur in the maize plant. The most noticeable and common changes that result under low-temperature stress in maize plants are the following: leaf wilting and chlorosis, root damage to system architecture, reduced surface area and volume of root and reduced root biomass [36,37]. During the seedling stage, cold stress also negatively impacts plant height [1].

Cold stress can also disrupt the normal functions at physiological level. A suboptimal temperature during the early growth phase of maize leads to a decrease in chlorophyll and carotenoid content, so assimilation pigments may be a valuable marker for identifying the most tolerant maize variants during the cold stress period [38,39,40]. Thermal stress can also have negative effects on several physiological parameters; for instance, the relative water content (RWC) can be damaged [41]. Under low-temperature stress, the RWC may decrease, causing premature leaf senescence [42]. The cold test (CT) is widely used in maize to forecast field performance at early sowing, providing an important marker in identifying variants with increased resistance to cold stress [43,44].

The mechanism of cold tolerance is complex and involves a variety of molecular pathways to provide plants with the ability to withstand cold stress periods. One of the most important plant hormones is abscisic acid (ABA), which plays a diverse role in physiological processes, from germination and seed dormancy to plant growth and development under severe environmental conditions [45,46]. The amount of ABA can be modulated in two different ways, de novo biosynthesis and catabolism. Through the second process, the level of ABA hormone can be reduced by ABA8′-hydroxylase, a cytochrome P450, which can hydroxylate the 8′-position of abscisic acid [47]. Thus, an increased expression of the ABA8′H gene can be correlated with a decreased quantity of ABA phytohormone. PP2C gene expression is controlled by the ABA-dependent pathway [48], and concomitantly, this gene is involved in numerous signaling pathways activated by abiotic stresses such as cold, drought, salt, wounding or by plant hormones (abscisic and gibberellic acids) [49]. Dehydration-responsive element-binding (DREB) transcription factors are involved in many stress responses in plants. A total of 65 DREB family gene members were identified in Zea mays L. [50]. The DREB1 gene has been found to increase cold tolerance in maize due to its capacity to bind directly to the raffinose synthase promoter, enhancing its expression, leading to raffinose biosynthesis [51]; moreover, raffinose accumulation is directly correlated with plant cold stress tolerance [52]. WRKY is a large family of genes, and various studies have established that WRKY transcription factors are involved in abiotic stresses response, such as cold, drought or salt, in different plant species [53]. In plants, the POR (protochlorophyllide oxidoreductase) gene plays a key role in the reactions chain, leading to chlorophyll generation. Thus, it catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria, followed by conversion to chlorophyll [54]. The POR expression gene is correlated with chlorophyll content [55]. This study analyzed the expression of the MYC transcription factor, which has many implications in plant physiological processes. MYC transcription factors are involved in abiotic stress signaling, being controlled by an ABA-dependent pathway [56]. In maize, the CCD1 (carotenoid cleavage dioxygenase 1) gene encodes an enzyme that has the ability to catabolize carotenoids [57]. It has been demonstrated that, in maize, the CCD1 enzyme can cleave a larger spectrum of substrates, such as lycopene, β-carotene or zeaxanthin [58].

Cold stress is a limiting factor for maize crop production, and it is essential for food security to determine genotypes with enhanced resistance to cold stress. The aim of the present research was to investigate the effects of plasma-activated water generated by dielectric barrier discharge on maize seeds and young seedlings grown at specific low temperatures. While most studies analyze direct plasma treatment and its effects on germination and growth-related parameters, the current research focuses on irrigation with PAW and also explores its effects at a molecular level by analyzing cold-response genes in defined landraces; these elements represent specific contributions to the field and serve to enrich the international databases. Three different experiments were carried out, in which two maize landraces were subjected to differential treatments. Subsequently, molecular, morphological, physiological and agronomic analyses were undertaken to find out whether plasma-activated water can mitigate cold stress in maize and whether there are different responses to cold in samples from control and stressed plots. This study focuses on maize landraces variations from the Plant Genetic Resources Bank Romania collection, which may be a valuable resource pool for breeding programs, aiming to create hybrids characterized by superior cold tolerance.

2. Materials and Methods

2.1. Plant Material and Experimental Conditions

Two Zea mays L. landraces with different resistance to cold stress were obtained from the Plant Genetic Resources Bank Suceava, Romania collection. The low-temperature-sensitive genotype, SVGB-11742, and the low-temperature-resistant genotype, SVGB-718, were instituted in three distinct experiments with different growth treatments. More information about the maize samples can be found in the Supplementary Materials (Table S1).

For the first experiment, in vitro seed germination, twenty-five seeds per lot of the two maize landraces (SVGB-718 and SVGB-1742), which were first surface-sterilized with a 1% sodium hypochlorite solution, were subjected to distinct growth treatments. The between-paper method was selected for in vitro seed germination using square growth plates (12 cm side). Initially, the seeds were split into two groups, one treated with plasma-activated water and one watered with untreated water (SVGB-718 with and without plasma-activated water treatment and SVGB-11742 with and without plasma-activated water treatment). All samples were maintained in the germinator chamber for seven days at a specific temperature (cycling process of two temperatures, i.e., 8 h at 20 °C and 16 h at 30 °C) and relative humidity (80%). Subsequently, samples were exposed to low temperatures for seven days as follows: cycling process of two temperatures, 8 h at 4 °C and 16 h at 9 °C. Meanwhile, the germinated seeds were watered with plasma-activated water and untreated water, with treatment specific to each lot. Finally, all growth plates were subjected to standard growth temperature (8 h at 20 °C and 16 h at 30 °C) for another seven days.

The second experiment was performed in a greenhouse of the Plant Genetic Resources Bank Suceava, Romania, under managed conditions, with 1500 luxes, 22 °C temperature, 65% relative humidity and with alternating light–darkness for 16 h and 8 h, for 14 days. For this experiment, the same landraces were exploited, SVGB-718 and SVGB-11742, which were watered with plasma-activated water and untreated water (which generated two experimental lots for each genotype). The Zea mays L. seeds were germinated in growth seedling pots (8 cm × 8 cm × 20 cm) containing a soil/vermiculite/perlite (2/1/1 w/w/w) substrate mixture. Each growth seedling pot included a single maize plant. After 14 days, during which the samples were maintained under normal growing conditions, the samples were divided into two groups, seedlings maintained at normal growing temperature and seedlings at low temperature. Finally, four experimental plots for each maize genotype were obtained, i.e., 1—normal temperature (NT) and untreated water (UW); 2—normal temperature (NT) and plasma-activated water (PAW); 3—low temperature (LT) and untreated water (UW); 4—low temperature (LT) and plasma-activated water (PAW). The experimental groups were subjected to cold stress for 7 days (cyclic process of two temperatures, 8 h at 4 °C and 16 h at 9 °C) and then kept for another 7 days in the greenhouse alongside the control lots. The maize seedlings were irrigated every day with 10 mL of plasma-activated water and untreated water specifically for each plot. The groups consisted of three biological replicates (three plants/lot).

The third experiment was realized in an experimental field in 2024. Initially, seeds of the SVGB-718 and SVGB-11742 variants (fifty seeds/lot) were soaked for 12 h in plasma-activated water and untreated water. Subsequently, the seeds were placed in the field following agronomic standards: 2 rows for each lot (25 seeds per row), 70 cm between rows, 25 cm between plants in the row and 5–7 cm sowing depth. Samples were sown on May 14 and harvested on 14 October 2024.

The experiments were conducted using a factorial design based on a completely randomized design (CRD) with three factors: genotype (SVGB-718, SVGB-11742), water treatment (untreated water, plasma-activated water) and temperature (normal, low). Each genotype was subjected to all combinations of water treatment and temperature. Statistical analyses were performed to assess the effects of the experimental factors on the measured traits.

2.2. Generation of Plasma-Activated Water (PAW)

Distilled water was activated using dielectric barrier discharge (DBD) equipment (Advanced Plasma Solution, Philadelphia, PA, USA). Experiments were performed at ambient air temperature (20–25 °C) and atmospheric pressure. A 20 mL volume of distilled water, placed into a Petri dish (8 cm diameter) and directly connected to the ground electrode, was exposed to plasma for 4 min. Between the water interface and the dielectric electrode was a distance of 4 mm, and the water layer thickness was 0.4 cm. The technical specifications of the DBD power supply were configured as follows: power: 300 W, voltage: 22 kV and duty cycle: 75%.

When producing PAW at atmospheric pressure in ambient air, external factors (such as temperature, humidity) can modulate the PAW’s physical and chemical properties. To avoid differences in the PAW’s characteristics, enough PAW was produced in one day and immediately stored in plastic tubes at −80 °C until use. When needed, PAW was defrosted and used to irrigate seeds/seedlings.

2.3. Measurement of Physical Parameters of Plasma-Activated Water

Certain physical parameters of the plasma-activated water were monitored, such as pH (FiveGo, Mettler Toledo, Shanghai, China), total dissolved solids (FiveEasy, Mettler Toledo, Shanghai, China) and conductivity (FiveEasy, Mettler Toledo, Shanghai, China). The results of the measurements are summarized in Table S2.

2.4. Germinated Seed Analysis

For all germinated seeds, from the first experiment, the radicle length was determined by a digital caliper. The measurements were conducted on days 7, 14 and 21.

2.5. Morpho-Agronomic Parameters Analysis

Various morpho-agronomic parameters were evaluated for the maize samples in the experimental field, such as growth vigor, cold resistance, silking time, flowering time, breaking plants, plant fall, number of sterile plants, the Fusarium resistance, the weight of 1000 seeds, plant height, main stipule insertion height, total number of leaves, number of leaves up to main stipule, stipule length, maximum stipule diameter, minimum diameter of the stipule, number of grains rows, number of grains/rows, grain length, grain width and grain thickness.

It is important to mention that all these parameters were strongly influenced by the meteorological conditions registered between 14 May 2024 and 14 October 2024. On 2 July, an intense storm occurred, with the wind speed reaching 17 m/s and a recorded precipitation amount of 8 mm/m², creating extreme conditions that severely affected crop stability and development (

Table 1. Values for temperature, rainfall and wind speed recorded on several days in July, with particular relevance to July 2, when a storm occurred and affected maize plants (storm dates are marked in yellow).

July	Temperature (°C)			Rainfall, Total (mm/m2)	Wind Speed (m/s)
	Mean	High	Low		Average	Maximum
1	26.5	34.1	18.8	0.0	1.6	8.5
2	21	28.8	15.2	8.0	1.0	17.0
3	13.9	15.3	12.3	3.6	1.8	8.5
30	20.2	25.1	16.2	0.0	1.9	10.7
31	21.7	28.7	13.8	0.0	0.8	7.2

). In addition, the storm was accompanied by large hailstones that further damaged the cultivated plants. Values for temperature, rainfall and wind speed over the entire period (14 May–14 October) are provided in the Supplementary Materials (Table S3), along with complete values recorded in July (Table S4).

2.6. Morphological Analysis

To identify the cold-resistant maize genotypes irrigated with treated and untreated water, some morphological characteristics of fresh seedlings from each lot were assessed. This included measurement of total plant, shoot and root length. Morphological analysis of the seedlings was undertaken on the last day of the second experiment (day 28).

2.7. Physiological Parameters Analysis

The relative water content (RWC) of fresh leaves of maize seedlings was determined according to the method described by Weatherley [59]. Fresh leaves from each plant (from the second experiment), grown under different conditions, were weighed to obtain their fresh biomass (FB). Then, the leaves were soaked in distilled water for 12 h at 4 °C to find the turgor weight (TW). Subsequently, the same leaves were introduced in a forced-air-convection drying oven at 60 °C for 72 h, and dry biomass was weighed. The values resulting from the measurements were applied in the following formula: RWC (%) = [(FW − DW)/TW − DW)] × 100, where DW = dry weight after oven-drying and TW = turgid weight.

Following the method described by Sumanta [60], the chlorophyll from fresh leaves of maize seedlings (from the second experiment) was isolated. With 10 mL of acetone, which was used as an extractant solvent, 0.5 g samples of leaf were homogenized. The obtained mixture was placed in 15 mL tubes and centrifuged at 4 °C and 10,000 rpm for 15 min. Next, 0.5 mL of supernatant was separated and mixed with 4.5 mL of acetone, followed by analysis of chlorophyll a, b and carotenoid content, using a UV–VIS spectrophotometer (PG Instruments T70 UV/VIS Spectrophotometer, Wibtoft, UK). Absorbance was measured at 665.2 nm, 652.4 nm and 470 nm. The achieved values were used in the following equations to determine the chlorophyll a, b and carotenoid concentrations (in µg/mL): Ch-a = 16.72 × A_665.2 − 9.16 × A_652.4; Ch-b = 34.09 × A_652.4 − 15.28 × A_665.2; Car = (1000 × A₄₇₀ − 1.63 × C_a − 104.96 × C_b)/221. These measurements were conducted for three replicates from all experimental lots.

In maize, the cold test is commonly used to assess seed quality in order to select seed lots for sowing in severe climates and to create new maize hybrids that have improved stand establishment. For the cold test, two experimental groups were established for each sample, the cold tolerance test group and the control group. Twenty-five seeds were sown in individual pots, which were maintained under normal conditions (22 °C), with alternating light and dark, for 14 days until the third leaf developed. Then, samples for the cold tolerance assay were placed in a climate chamber under controlled conditions for 7 days, as follows: 8 h at 4 °C and 16 h at 9 °C. Finally, the samples were transferred for another 7 days with the control samples under normal growing conditions. At last, 20 plants from each lot were selected and dried in a forced-air-convection drying oven at 180 °C until the samples had a constant weight. The cold test index was evaluated according to the accumulation of dry mass, expressed as an index (Ki) calculated as the ratio of the dry mass of the cold-stressed sample to that of the control sample, according to the following scale: resistant genotypes (>80%); semi-resistant genotypes (60–79%); low-resistance genotypes (40–59%); susceptible genotype (<40).

2.8. RNA Isolation and Quantification

RNA extraction was performed using 30 mg of leaf and root tissue (from second experiment), stored in RNA Save at −80 °C, using the SV Total RNA Isolation System (Promega, Madison, WI, USA) and following the manufacturer’s instructions. To determine the concentration and purity of total RNA samples, a NanoDrop One UV–VIS (Thermo Scientific GmbH, Dreieich, Germany) spectrophotometer was exploited.

2.9. Relative Gene Expression Analysis

The GoTaq^® 1-Step RT-qPCR reagent system (Promega, Madison, WI, USA) was used for cDNA synthesis and gene expression analysis, including specific primer pairs for each of the seven genes assayed (Supplementary Table S5) and following the manufacturer’s instructions. The CFX96 Touch Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA) was exploited for the amplification reactions, with the following steps: reverse transcription reaction at 37 °C for 15 min, followed by inactivation of reverse transcriptase and activation of GoTaq DNA polymerase at 95 °C for 10 min. The third step, replicated 40 times, included the following steps: 95 °C for 10 s (denaturation), 60 °C for 30 s (annealing and data collection) and 72 °C for 30 s (extension). A run-end step for melting curve analysis was included. For seven genes (Table S5), the relative expression was analyzed from root and leaf tissues to identify the response of maize seedlings grown under different conditions. The relative expression of each gene for the experimental variants was inferred based on a reference housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and predicted according to Livak using the ΔΔCt algorithm [61]. All reactions were carried out in two technical replicates for all three biological replicates.

2.10. Statistical Data Analysis

Prior to analysis, assumptions of normality and homogeneity of variance were tested using the Shapiro–Wilk and Levene tests. Data that met assumptions were analyzed without transformation, while count data (total number of leaves, number of leaves up to the main stipule, number of grain rows and number of grains per row) were square-root-transformed when assumptions were not met. Percentage data (cold test index and relative water content) were arcsine-square-root-transformed. Chlorophyll and carotenoid concentrations were log-transformed where necessary. Gene expression levels were analyzed on log2-transformed values (ΔCt). For field experiments, a one-way ANOVA test was used, while for laboratory experiments, a two-way ANOVA was conducted to analyze the effects of genotype and treatment, including their interaction. When significant effects were detected, pairwise comparisons were conducted using Tukey’s HSD post hoc test (p < 0.05). All analyses were performed in GraphPad Prism 9.

3. Results

3.1. Germinated Seed Analysis of Maize Samples Exposed to Cold Stress and Watered with PAW

The effects of cold can be inferred by analyzing the morphological traits of germinated seeds for both lots, watered with normal and treated water (PAW) and exposed to different thermal regimes. The recorded data (

Table 2. Summary statistics on radicle length (mm) of two maize samples grown under different conditions.

Measurement Day	SVGB-718		SVGB-11742
	UW	PAW	UW	PAW
	Avg ± SD	Avg ± SD	Avg ± SD	Avg ± SD
Day 7	55.29 ± 23.74	37.92 ± 17.41	32.83 ± 15.05	49.79 ± 29.89
Day 14	85.99 ± 42.34	62.55 ± 25.78	74.74 ± 25.33	78.23 ± 35.79
Day 21	100.39 ± 42.92	80.44 ± 26.44	84.31 ± 28.20	93.16 ± 36.7

Avg = average, SD = standard deviation, UW = untreated water, PAW = plasma-activated water.

) showed that at the first germination stage, when the seeds were placed at a specific temperature, the average radicle length was higher for SVGB-718 watered with untreated water (55.29 mm) compared to the lot irrigated with PAW (37.93 mm). Instead, SVGB-11742 registered a superior mean radicle length value for seeds watered with PAW (49.79 mm). Subsequent exposure of germinated seeds for seven days to cold stress revealed superior development for SVGB-718 irrigated with untreated water (85.99 mm) than the lot irrigated with treated water (62.55 mm). Different data were obtained for the SVGB-11742 sample when cold stress led to a superior development of the treated lot (PAW lot). To assess the samples’ ability to recover after a stress period, germinated seeds were placed for another seven days at a specific temperature. Superior development was recorded for PAW-irrigated SVGB-11742 and UW-irrigated SVGB-718.

3.2. Morpho-Agronomic Parameter Analysis of Maize Samples Exposed to Untreated Water and Plasma-Activated Water and Grown in Field Conditions

The treatment with PAW stimulated an increase in the growth rate of plants in the experimental field. This was confirmed by the shortening of the silking and flowering time (

Table 3. Morpho-agronomic parameter data of two maize samples from the experimental field.

Morpho-Agronomic Parameters	SVGB-718 (SUW)	SVGB-718 (SPAW)	SVGB-11742 (SUW)	SVGB-11742 (SPAW)
Growth vigor (3/5/7)	5	5	7	7
Cold resistance (3/5/7/9)	5	5	7	7
Silking time (days after sowing)	59	57	64	64
Flowering time (days after sowing)	58	56	63	64
Breaking plants (%)	31	0	18	24
Plant fall (%)	38	85	9	11
Number of sterile plants	6	7	11	14
Fusarium resistance (FAO indicators)	5	4	3	4
Weight of 100 seeds (g)	180	220	300	300

SUW = soaked in untreated water; SPAW = soaked in plasma-activated water; growth vigor—3 = weak, 5 = intermediary, 7 = vigorous; cold resistance—3 = sensitive, 5 = intermediate, 7 = resistant, 9 = very resistant.

) for the SVGB-718 maize sample. The breaking phenomenon was not reported for the SVGB-718 plants that had been treated with PAW, compared to the high percentage reported for the initially untreated plants (31%). A 22% higher value of 1000 seed mass after harvest was reported for the PAW-treated lot of SVGB-718. Resistance to the Fusarium pathogen increased for the plasma-activated-water-treated SVGB-718 maize sample. Meanwhile, plasma treatment did not improve cold resistance or the percentage of fallen plants. No significant differences were reported between the PAW-treated and untreated groups for the SVGB-11742 variant, apart from the percentage of broken and fallen plants and the number of sterile plants, where plasma-activated water did not improve these values. The experiment began with 25 individuals/group, but because of the storm, the numbers reduced to n = 15 SVGB-718 UW, n = 18 SVGB-718 PAW, n = 16 SVGB-11742 UW and n = 14 SVGB-11742 PAW.

PAW treatment led to an improvement in the values of some morphological parameters in the SVGB-718 genotype, such as plant height, total number of leaves, stipule length, maximum stipule diameter, number of grains rows, number of grains per row, grain length and grain thickness (Figure 1A,C,E,F,H,J,L). With regard to the SVGB-11742 landrace, the measurements from the field experiments did not reveal significant differences between both groups (with and without PAW), apart from plant height, total number of leaves and grain thickness (Figure 1A,C,L), where the treatment with plasma-activated water led to higher values.

3.3. Morphological Analysis of Maize Samples Exposed to Cold Stress and Watered with PAW

Morphological data revealed different results according to the evaluated genotypes. Thus, PAW treatment for SVGB-11742 improved the development of plants grown at normal and low temperatures, with superior development of these plants being reported (

Table 4. Summary statistics of morphological parameters of two maize samples cultivated under different conditions (n = 3).

SVGB-718
	NT + UW	NT + PAW	LT + UW	LT + PAW
	Avg ± SD	Avg ± SD	Avg ± SD	Avg ± SD
Root length	24.79 ± 5.42	23.88 ± 6.24	17.38 ± 6.12	14.5 ± 7.71
Shoot length	40.24 ± 5.64	37.45 ± 10.29	27.38 ± 7.55	23.43 ± 4.48
Plant total length	65.03 ± 7.47	61.33 ± 13.56	44.77 ± 11.23	37.93 ± 8.87
SVGB-11742
	NT + UW	NT + PAW	LT + UW	LT + PAW
	Avg ± SD	Avg ± SD	Avg ± SD	Avg ± SD
Root length	26.67 ± 9.09	27.85 ± 8.41	23.48 ± 6.86	26.2 ± 8.94
Shoot length	44 ± 6.82	49.9 ± 8.34	38.86 ± 6.81	32.65 ± 7.3
Plant total length	70.67 ± 12.7	77.75 ± 11.72	62.33 ± 11.52	58.85 ± 14.21

Avg = average, SD = standard deviation, UW = untreated water, PAW = plasma-activated water, NT = normal temperature, LT = low temperature.

). Different results were noticed for SVGB-718, where PAW treatment did not enhance the growth of maize plants exposed to normal and low temperatures.

3.4. Physiological Parameter Analysis of Maize Samples Exposed to Cold Stress and Watered with Untreated Water and Plasma-Activated Water

Cold stress is known to decrease the levels of assimilatory pigments such as chlorophylls and carotenoids, which make them important indicators for selecting maize genotypes more resistant to low temperatures. No statistically significant differences were found in assimilatory pigment concentrations for most conditions, although an increasing trend in carotenoids was observed for cold-stressed SVGB-718 plants treated with PAW (Figure 2C). The results in Figure 2 show that PAW treatment did not improve the concentration of chlorophyll pigments and carotenoid compounds in leaves under cold stress conditions, apart from the carotenoid content of the SVGB-718 sample (Figure 2C). The chlorophyll a concentration of SVGB-11742 plants (Figure 2D) subjected to cold and irrigated with PAW was 25.64% above that of SVGB-718 (Figure 2A), respectively, and 24% higher for chlorophyll b (Figure 2B,E).

To assess the adaptability of the two maize landraces to cold stress, the relative water content was measured (Figure 3). The results showed that the relative water content value was higher for SVGB-718 exposed to cold and irrigated with PAW compared to plants irrigated with untreated water. Differently, for SVGB-11742, PAW did not enhance the relative water content level of the plants grown under low-temperature conditions; furthermore, the value was quite similar to that of the irrigated lot with untreated water.

The cold test is an index that can provide important data on the ability of maize to cope with cold stress. Thus, PAW treatment did not improve plant resistance to low temperatures compared to control lots of both evaluated maize genotypes; moreover, the cold test index value decreased for the lots irrigated with plasma-activated water (

Table 5. The cold test index value for SVGB-718 and SVGB-11742 maize samples watered with untreated water and plasma-activated water. UW = untreated water, PAW = plasma-activated water (n = 3).

	Cold-Test Index
SVGB-718	UW	56%
	PAW	36%
SVGB-11742	UW	77%
	PAW	56%

).

3.5. Gene Expression Analysis of Maize Samples Exposed to Cold Stress and Watered with PAW

When plants are subjected to stress, various mechanisms are triggered to confer plant resistance to difficult environmental conditions. Several changes can occur at the molecular level, including the expression of genes related to growth, development and response to abiotic stresses. In the current study, the relative expression of the ABA8′H gene, related to signal transduction, was evaluated. The effects of cold stress on ABA8′H gene expression were different depending on the examined tissue (leaves/roots) and the water treatment applied (Figure 4a). In general, ABA8′H gene expression was approximately equal in both tissues for the maize samples, apart from plants in the LT + PAW lot of the SVGB-781 genotype. The highest level of ABA8′H gene expression was reported for SVGB-718 in the root tissue in LT + PAW, while the lowest expression was observed for the similar tissue and genotype in the NT + UW lot. Other data concern the expression of the CDD1 gene, which is involved in plant defense. The relative expression was superior in leaf tissue compared to roots for both analyzed maize samples (Figure 4b). In leaf (SVGB-11742 genotype) and root tissue (SVGB-718 genotype), the relative expression was upregulated for the LT + PAW groups. Instead, expression of the CDD1 gene was downregulated in leaf tissue for SVGB-718, respectively, and in root tissue for the SVGB-11742 sample.

The results shown in Figure 5a demonstrate the variations in the expression levels of the gene encoding DREB1. After subjecting the plants to cold stress and watering with plasma-activated water, DREB1 gene expression was upregulated in the leaf and root tissues of SVGB-718. The DREB1 gene was differentially expressed in the SVGB-11742 maize genotype. In the leaves, the gene expression decreased in the LT + PAW lot compared to the NT + PAW lot but was upregulated compared to the LT + UW and NT + UW lots. Similar results were found in the root tissue of the SVGB-11742 sample. Different temperatures and types of water used for plant irrigation induced variations in MYC gene expression (Figure 5b). Therefore, in the SVGB-718 maize sample, the expression level was decreased for the LT + PAW lot compared to the other three groups (NT + UW, LT + UW and NT + PAW) in the leaf tissue, and the MYC gene was overexpressed for the LT + PAW group in the root tissue. On the other hand, for the SVGB-11742 variant, the relative expression of the MYC gene was increased in the LT + PAW lot in the leaf tissue. In the second evaluated tissue, there was slightly increased expression in the LT + PAW group compared to the plants irrigated with normal water and grown at low and normal temperatures, but there was a decrease compared to the plants irrigated with PAW and grown at normal temperature.

The relative expression of the POR gene in the plants grown at normal temperature and irrigated with plasma-activated water was upregulated in the leaf tissue for both maize landraces, respectively, and in the root tissue for SVGB-11742. Instead, the POR gene was overexpressed in the plant roots grown at low temperature and treated with plasma-activated water (Figure 6a). Relative expression measurements showed that PP2C expression was differentially increased in the leaf tissue. Therefore, in SVGB-718, higher expression was reported for the plants maintained at normal temperature and watered with PAW, while in SVGB-11742, elevated PP2C expression was related to the plants maintained at low temperature and irrigated with plasma-activated water (Figure 6b). The data showed that PP2C expression in the root tissue was completely distinct, overexpressed in the LT + PAW group of the SVGB-718 variant and higher in the plants irrigated with plasma-activated water and grown at normal temperature for the SVGB-11742 genotype. The WRKY transcription factor was significantly overexpressed in the leaf tissue of the plants exposed to normal temperatures and irrigated with untreated water compared to the other three groups of the SVGB-718 sample (LT + UW, NT + PAW, LT + PAW) (Figure 6c). The abiotic stress and treatment with plasma-activated water increased the relative expression of the WRKY gene in the root tissue of the SVGB-718 maize variant. The WRKY gene expression was higher in the root tissue compared to leaves as regards the SVGB-718 landrace. The reported data show that the WRKY gene was expressed higher in the leaves of plants of the SVGB-11742 variant exposed to low temperatures and irrigated with untreated water, while in the roots of plants subjected to abiotic stress and treated with PAW, a higher expression of the WRKY gene was reported.

4. Discussion

In recent decades, concern about the climate has become an important topic for many research groups. The agricultural sector is the most affected, where variations in temperature, rainfall regimes and extreme weather events can disrupt the normal development of plant species and lead to difficulties in food security and agricultural outputs. Temperature is an important factor for plant growth and reproduction; therefore, extreme temperatures can negatively damage plant development and, subsequently, crop yields, with major economic losses [62,63,64]. Zea mays L. originates in tropical and subtropical regions, and even though maize has been successfully introduced in temperate zones, it has not been completely adapted to temperate climates, and low temperatures can threaten the species [37]. Plant species must constantly adapt to changing environmental conditions, such as cold stress; plants are well-adapted organisms that have developed multiple morphological, physiological and molecular survival mechanisms during their evolution [65]. However, new methods are being investigated to increase plant resistance to abiotic stress factors, and the treatment of seeds/seedlings directly/indirectly with cold plasma is a method of interest in current research [66]. In this work, the molecular, physiological and morphological responses of two maize landraces exposed to cold stress and treated with plasma-activated water were analyzed.

Sub-optimal temperatures delay maize seed germination. Several studies have demonstrated that cold stress maintains seed dormancy, negatively regulates the germination process and reduces the germination rate [67,68,69]. On the other hand, research has shown that plasma treatment can improve the seed germination process in many plant species, including maize [70]. This phenomenon can be explained as an indirect effect of cold plasma on germination due to the high concentration of reactive oxygen and nitrogen produced during the plasma gas generation process. Therefore, RONS interferes with the ABA and GA pathways, with effects on redox balance and stimulating germination [8]. In the current study, the data revealed that the extension of germination time was different for the analyzed variants. Similar results were also reported in previous research [69,71]. Thus, SVGB-11742, when subjected to low temperature, responded more positively to plasma-activated water treatment compared to SVGB-718.

The effects of low temperatures on maize seedlings are variable, depending on the intensity of the stress, and they can range from leaf wilting or growth inhibition to plant death. At low temperatures, a decreased cell multiplication and growth [1], reduced light energy conversion efficiency [72] and inefficient water and nutrient absorption and management [73,74] were reported, which can lead to a decline in the morphological traits of seedlings and roots. Plasma-activated water can be applied as a sustainable treatment to enhance plant growth and development. Numerous studies have shown that the use of plasma-activated water has favorable effects in promoting the growth of plant species [75,76,77,78]. In this study, low temperatures were found to reduce the growth and development of both maize genotypes. However, plasma-activated water treatment had a favorable effect on root length in the cold-exposed variety SVGB-11742. PAW treatment also led to significant growth in the SVGB-11742 genotype grown at normal temperatures.

As a site of chlorophyll production and a fundamental plant cell organelle, chloroplasts play a critical role in photosynthesis process. During periods of abiotic stress, the accumulation of active oxygen species can damage chloroplasts, which can lead to a decreased uptake of assimilatory pigments such as chlorophyll or carotenoids [79]. Studies have revealed that the chlorophyll content can be elevated in leaves of plants irrigated with plasma-activated water [75,80]. Plasma-activated water treatment did not have the expected effects on the chlorophyll a and b contents of the SVGB-718 genotype subjected to low temperatures; instead, PAW treatment increased the carotenoid concentration. The content of chlorophyll and carotenoids was not improved following PAW treatment in the SVGB-11742 sample. Cold stress can lead to a decline in the relative leaf water content value of plant leaves by disrupting water transport and availability. This can have a negative impact by restricting photosynthesis, impairing physiological processes and stomatal closure [81]. The relative water content value was increased for SVGB-718 exposed to cold and watered with plasma-activated water compared to the lots irrigated with untreated water or grown at normal temperature. Different results were achieved for SVGB-11742, where PAW treatment did not have the predicted response. The cold test for maize can be used as a method to assess seed germination and seedling development under unfavorable environmental conditions. In the present study, plasma-activated water did not exhibit a favorable effect on the seedling development of both maize genotypes subjected to low temperatures. In other studies, it has been reported that the value of the cold test index varied among different genotypes [82].

Various stresses are often reported as challenges for plants. If vegetal organisms are subjected to difficult environmental conditions, various loci can mediate plant responses, with each locus exerting a slight effect. Thus, the morpho-physiological changes that occur in response to abiotic stresses represent the combined effect of hundreds of genes [83].

ABA (abscisic acid) is an important hormone with various implications in plant physiological processes such as adaptive response to abiotic and biotic stresses or growth and development under normal conditions [84]. On the other hand, the ABA8′H gene encodes ABA8′-hydroxylase, an enzyme that can break down abscisic acid, which is the main pathway for ABA catabolism in higher plants [85]. In the current work, gene expression was higher in the SVGB-718 root tissue of the PAW-irrigated and cold-stressed lot, not only compared to the control lot but also compared to the SVGB-11742 genotype. Therefore, this may be associated with a decrease in the amount of abscisic acid and subsequently with the resistance of the variety to less favorable environmental conditions. The ABA8′H gene was expressed less in the leaf tissue of SVGB-11742 in the PAW-watered and cold-exposed group but not less compared to the control. DREB (dehydration-responsive element binding) genes belong to the larger AP2/ERF transcription factor superfamily, with numerous functions in plant response to abiotic and biotic stresses [86]. During this research, the relative expression of the DREB gene in leaves and roots was quantified for the two different maize landraces. It was reported that the DREB gene was upregulated in leaves and roots of the PAW-irrigated and cold-stressed SVGB-718 plants. Instead, for the SVGB-11742 genotype, the DREB transcription factor was upregulated in the LT + PAW lot compared to plants maintained at normal growing temperatures and irrigated with untreated water, but it was downregulated compared to plants subjected to normal temperature and irrigated with PAW. The POR gene may be indirectly associated with growth and development processes in plants. The POR gene encodes protochlorophyllide oxidoreductase (NADPH), an enzyme essential for chlorophyll biosynthesis. During photomorphogenesis, the POR enzyme facilitates the conversion of protochlorophyllide a to chlorophyllide a in the presence of light. Chlorophyllide is further processed into chlorophyll [87]. Plasma-activated water had a favorable influence on POR gene expression in the roots of cold-exposed SVGB-718 plants. Also, in the leaf tissue, its expression was higher in plants treated with plasma-activated water compared to those irrigated with untreated water and subjected to cold. Regarding the SVGB-11742 genotype, treatment with PAW had a positive effect on plants grown at normal temperature, with the POR gene being overexpressed in the normal-temperature-exposed and PAW-irrigated groups compared to the other lots, both in leaf and root tissues. MYC (Myelocytomatosis) transcription factors are controlled by the JA pathway, playing a crucial role in plant growth and development, and they are also involved in the response to environmental stress [88]. MYC genes are key players in plant cold tolerance, with their function influencing the expression of genes that enable plants to cope with cold tolerance [89]. In the present study, plasma-activated water treatment had a favorable effect on MYC gene expression, which was overexpressed in the leaf and root tissue of the SVGB-11742 and SVGB-718 plants, respectively, that were exposed to cold stress. Thus, the plasma-activated water enabled the plants to survive and develop in less favorable environmental situations. The PP2C (2C protein phosphatase) gene acts as a negative regulator of abscisic acid signaling in plants. The PP2C gene has an important role in suppressing ABA signaling by interfering and inhibiting SnRK2 kinases, which are positive upregulators of the ABA response [48]. The PP2C gene was overexpressed in the root tissue of the PAW-treated and cold-exposed SVGB-718 plants. Similarly, an increase in PP2C gene expression was reported in the leaf tissue of the PAW-treated plants of the SVGB-11742 genotype treated with PAW and maintained at low temperature compared to the other groups. For the PAW-irrigated plants exposed to cold, the lowest PP2C gene expression was recorded in the leaf tissue of the SVGB-718 genotype. It can also be affirmed that the relative expression of the gene was higher in the root tissue compared to the leaf tissue. The CDD1 (constitutive defense without defect in growth and development 1) gene encodes a protein that manages plant defense mechanisms. The CDD1 mutation leads to an increased defense response in plants through constitutive activation of salicylic acid (SA) signaling pathways, which are crucial for plant immunity [90]. The relative expression of the CDD1 gene was higher in the leaf tissue of the SVGB-11742 plants from the PAW-irrigated and low-temperature-exposed group. Specifically, PAW treatment had a favorable role in increasing cold stress tolerance in the SVGB-11742 genotype. Moreover, at the root tissue level, a higher expression was reported for the SVGB-718 maize landrace irrigated with PAW and subjected to low temperature. WRKY transcription factors play a crucial role in plant stress response. They are triggered by various abiotic stresses and regulate both ABA-dependent and ABA-independent signaling pathways, impacting how plants cope with environmental challenges [91]. In the current study, the obtained data showed that the WRKY gene was upregulated in the leaf tissue of both the SVGB-718 and SVGB-11742 maize landraces treated with plasma-activated water and exposed to cold stress. Also, in the leaf tissue, gene expression was downregulated in the plants from the low-temperature-exposed and PAW-irrigated lots compared to the other experimental groups for both the SVGB-718 and SVGB-11742 genotypes. Even if the molecular response showed several changes caused by the PAW treatment, there are plenty of supplementary studies which can be conducted to obtain a more comprehensive idea about the changes that occur in the seed. Among these, we can mention an analysis which focuses on the variation in the seed microbiome, as early growth and stress response may be mediated not only by direct physiological effects of PAW on plants but also by alterations in the microbiome. Most studies analyze the effects on the microorganism population after direct treatment of the seed [27,92,93], but indirect treatments are also of interest. Data collection from the experimental field showed that plasma-activated water had different effects on the development of the two maize genotypes. As previously mentioned, there were some extreme weather events that affected the normal development of the plants and, consequently, the results obtained at this stage. Thus, the agronomic analyses were influenced not only by the experimental conditions established in this study but also by uncontrolled factors, and it is recommended to repeat this assessment in future research using a more rigorous design.

This study highlights the relevance of plasma-activated water in enhancing plant resistance to thermal stress. The findings represent a valuable contribution to the field and could be further strengthened through more comprehensive studies involving larger sample sizes as well as replication and refinement of the experimental design.

5. Conclusions

Maize (Zea mays L.) is a major global cereal that suffers yield and quality losses under low-temperature stress, making sustainable strategies to enhance its resilience increasingly important. Non-thermal plasma, which can be used to generate plasma-activated water (PAW), offers a promising eco-friendly approach to support plant stress tolerance. In this study, two maize genotypes with different cold resistances (SVGB-11742, highly resistant; SVGB-718, moderately resistant) were exposed to combinations of untreated or plasma-activated water and normal or low temperatures.

The results indicate that PAW can improve growth under optimal temperature conditions, but its effects under low-temperature stress were limited, with little impact on seedling development or leaf pigment content. Interestingly, PAW appeared to influence molecular responses more than morphological traits, particularly enhancing stress-related gene expression in the moderately resistant genotype, suggesting that its primary role may be as a priming agent rather than a direct growth stimulant under the conditions tested. While environmental constraints and low replication limited the consistency of the observable effects, these findings provide preliminary evidence that PAW can modulate stress responses at the molecular level, highlighting its potential for improving maize resilience. Future research should explore whether these molecular changes translate into longer-term agronomic benefits under field conditions.