Plasma-Mediated Regulation of Environmental Stresses in Plants and Animals

1. Introduction

With global climate change intensifying and environmental pollution increasing, biological systems are facing unprecedented environmental pressures[1]. These pressures significantly affect the stability of agricultural production systems, including animal husbandry and crop cultivation[2]. Crops plants are susceptible to a variety of environmental stresses during their growth and development, and unfavorable environments are the main cause of reduced crop yields[3]. Surveys indicate that a 1% increase in agricultural yield translates to a 0.6–1.2% decrease in the absolute number of poor households worldwide[4]. In addition, animal husbandry has been seriously affected by climate change and environmental degradation in recent years. In industrial feeding environments, exposure to ammonia increases the probability of respiratory disease in animals by 15–20%[5]. Heavy metal contaminants can be enriched through the food chain, ultimately affecting meat safety[6]. The presence of pathogenic microorganisms will also increase the prevalence of animals and affect their growth and reproduction[7]. Heat stress causes dairy cows to experience elevated body temperatures, which can impact their reproductive performance, including hormonal imbalances, reduced oocyte and semen quality, and decreased embryo development and survival[8]. A study on European farms found that heat stress can cause a 2.8% decrease in milk production and a 5.4% loss in monthly income[8]. Meanwhile, the global population is predicted to grow to 9.7 billion by 2050, necessitating an increase of approximately 70% in food production to meet the demands of this population[4]. Reduced production of crops such as wheat, corn and rice and damage to livestock industry could have serious consequences, including exacerbating global food shortages, poverty, and hunger.

Technical innovations are needed to overcome recent crises in agricultural practices. Atmospheric pressure non-thermal plasma (APNTP) technology developed in physics has received attention in recent years as a potential tool for fostering adaptations to changes in climate and agricultural environments. Plasma is an ionized gas, often referred to as the fourth state of matter, alongside solids, liquids and gases[9,10]. In the plasma state, gas molecules are ionized, producing a mixture of positively charged ions and free electrons, along with neutral particles[11]. Plasmas can be divided into various types based on their temperature, density, and specific properties, including thermal, non-thermal or cold, high-density, and low-pressure plasmas[12]. Among these, APNTP is considered the most suitable type for biological applications due to their ability to generate and sustain plasma at atmospheric pressure[13]. The overall gas temperature remains close to ambient temperature, despite the high energy of the electrons in APNTP[14]. Various types of APNTP are being actively researched for their industrial applications (Figure 1).

To date, APNTP has been used in medicine and agriculture. Medical applications include sterilization of medicine materials and human living tissues, assisting blood coagulation, chronic foot, leg ulcers, wound healing and tissue regeneration[22,23,24]. Plasma also shows positive effects on the growth, development and crop yield[25,26,27]. Many plant seeds are subjected to a pre-sowing plasma treatment to increase the likelihood of timely and uniform germination, including radish (Raphanus sativus), carrot (Daucus carota sativus L.), soybean (Glycine max), bean (Phaseolus vulgaris), mung bean (Vigna radiata), black gram (V. mungo), pea (Pisum sativum), lentil (Lens culinaris), peanut (Arachis hypogaea), alfalfa (Medicago sativa), and chickpea (Cicer aruetinum), blue lupine (Lupinus angustifolius), Egyptian clover (Trifolium alexandrinum), fenugreek (Trigonella foenum-graecum), and mimosa (Mimosa pudica, M. caesalpiniafolia)[28,29]. For animal health, plasma treatment can achieve microbial sterilization efficiency of more than 90%[30], and a wound healing rate of more than 60%[31]. In addition, it can also improve the sperm motility of animals and improve reproductive efficiency[32]. Furthermore, due to its ability to effectively inactivate microorganisms and enzymes, APNTP can also be used as a disinfectant in food packaging, thereby preserving the freshness and extending the shelf life of food[33,34,35,36].

Currently, plasma has demonstrated its potential in fostering adaptations in plants and animals to climate and agricultural environmental changes. Although studies have shown that animals and plants treated with APNTP are more resistant to biological and abiotic stresses, the outcomes of these treatments vary depending on the animal and plant species, plasma devices, and environmental stresses, and the underlying regulatory mechanisms are still poorly understood[37]. In this review, we summarize and discuss case studies evaluating the improvement of animal and plant resistance through plasma under different environmental stresses and the relevant regulatory mechanisms to examine whether plasma technology can be a promising tool in solving environmental problems.

2. Plasma-Mediated Responses to Environmental Stress in Plants

Plasma agriculture is an emerging field in recent years, particularly in improving plant stress resistance[38]. Plant stress regulation involves the mechanisms and responses that plants employ to cope with adverse environmental conditions, ensuring their survival and productivity. Currently, environmental stresses that affect plant growth and development mainly include drought, salinity, extreme temperatures, nutritional deficiencies, metal toxicity, and biotic stresses such as viral, bacterial, and fungal infections (Figure 2). Studies have demonstrated that plasma can enhance the tolerance of plants to various environmental stresses through activating antioxidant systems, promoting expression of defense related genes, regulating plant hormonal and physiological responses, and signal transduction (Table 1).

2.1. Drought

Drought is an environmental stress that can significantly impact the growth and development of crops on farmland. Research has shown that drought can cause various physiological changes in plants, including stomatal closure, impaired photosynthesis, changes in leaf morphology, premature senescence, and changes in the root system due to insufficient water resources[66]. Notably, plasma may mitigate the negative effects of drought on plants by increasing seed germination and seedling growth. Atmospheric dielectric barrier discharge (DBD) plasma treatment was found to promote wheat seed germination and seedling growth under drought stress[40]. Compared with the "drought stress" group, the germination rate of the "plasma + drought stress" group increased by approximately 17.3%. The germination drought resistance index also increased by 15.1%, and the average root and shoot lengths increased by 20.0% and 31.9%, respectively[40]. Similar results also were obtained in wheat seeds treated with High Voltage Electrical Discharge (HVED). HVED pre-treatment can improve germination rate, germination index value, and shoot and root growth under drought conditions[41]. In addition, plasma also exhibited a promoting effect on the seed germination and seedling growth of oil seed rape, barley, alfalfa, and tomato under drought stress. The study found that capacitive coupled plasma (CCP) treatment significantly increased the germination rate of rapeseed to 6.25%, and significantly increased the dry weight, root length and number of lateral roots of rapeseed[39]. Plants grown from barley seeds treated with DBD plasma under drought conditions had stronger and denser root systems than control seeds, with a 20% increase in the number of roots and a 70% and 80% increase in the length and diameter of the taproot, respectively[42]. Phenotypic observations tomato seedlings under drought stress showed that seeds primed with cold plasma had higher drought resistance potential at the seedling stage[67]. alfalfa Seeds treated with 40w cold plasma/low-power CPT had higher germination potential, germination rate, seedling height, root length, and vigor index[68]. The mechanisms of plasma-induced drought resistance can be divided into three categories: 1) Improved germination rate—plasma treatment breaks seed dormancy and enhances water absorption capacity, thereby improving germination rate; 2) Improved root system—enhanced root development helps plants obtain deeper soil moisture and improve drought resistance; and 3) Enhanced physiological response—plasma treatment can induce physiological changes, thereby improving plant water use efficiency and stress response.

2.2. Salinity

Salinization is known to affect plant growth by decreasing soil fertility. Studies have shown that as salinity increases, the germination rate of seeds decreases significantly. Although plasma treatment did not affect plant size and root system under salt stress, plants grown from plasma-treated seeds had higher concentrations of chlorophyll a and b, while the concentration of proline decreased by approximately 50%[42]. In addition, high voltage electrical discharge (HVED) pretreatment can improve wheat drought and salt resistance. Under varying levels of NaCl salt stress, HVED treatment could change the structure of wheat seed surface, making it easier to absorb water, thereby improving germination rate, germination index value, and growth of aerial parts and roots[41]. Plants grown under salt stress accumulate more Na⁺ and less Ca⁺, leading to ion imbalance. Notably, APNTP treatment can alleviate this effect by reducing Na⁺ accumulation and increasing K⁺ and Ca⁺ levels in plant cells under salt stress[45]. APNTP also upregulated the activity of enzymes related to secondary metabolic assimilation, the accumulation of reactive oxygen species (ROS), and the content of malondialdehyde (MDA)[45]. In general, APNTP treatment cannot completely offset the effects of salt stress. However, in certain instances, APNTP enhances the germination rate and surface changes of seeds.

2.3. Extreme Temperature

Temperature fluctuations can have a significant impact on seed germination and subsequent plant growth, particularly in crops that are more susceptible to temperature-related stress. Extreme temperature fluctuations have the potential to result in a reduction in crop yield or, in severe cases, complete crop failure[69]. Rice (Oryza sativa L.) seeds showed delayed germination under heat stress, which was associated with the expression levels of abscisic acid (ABA, a germination-inhibiting hormone) and α-amylase (starch-hydrolyzing enzyme). However, seed germination rate was significantly restored after APNTP treatment[51]. Genes involved in ABA biosynthesis (OsNCED2 and OsNCED5) were downregulated, while genes involved in ABA catabolism (OsABA8′OH1 and OsABA8′OH3) and α-amylase genes (OsAmy1A, OsAmy1C, OsAmy3B, and OsAmy3E) were upregulated[51]. Similarly, the germination rate of plasma-treated seeds was higher even at low temperatures[42]. This suggests that plasma appears to improve plant tolerance to extreme temperatures.

2.4. Metal Toxicity

Some contaminated soils contain a variety of toxic heavy metal elements, which can accumulate in plants and eventually cause human poisoning[55]. In addition, these heavy metals can cause plant chlorosis, wilting, leaf curling, and slow plant growth[70]. Research has found that low-pressure dielectric barrier discharge (LPDBD) technology can alleviate the damage and growth retardation of wheat plants under cadmium (Cd) stress[53]. The mitigation effect of plasma on Cd toxicity may originate from the roots and is related to the conduction mechanism of NO[53]. Severe Cd stress leads to reduction in grain yield and monounsaturated fatty acid (MUFA) content in wheat. APNTP alleviates Cd stress by changing the physiological and biochemical characteristics and fatty acid composition of wheat, thereby optimizing wheat growth and yield[57]. In addition, plasma can play an auxiliary role in alleviating heavy metal stress. APNTP combined with foliar-applied selenium nanoparticles can regulate Cd toxicity by changing the essential oil content, chlorophyll a, chlorophyll b and total chlorophyll in sage[71]. Plasma-activated water appears to have a similar effect as shown in soybeans (Glycine max) in which treatment with plasma-activated water resulted in a five-fold reduction in the uptake of heavy metals (Pb)[58]. Maize (Zea mays L.) treated with plasma-activated water responded to arsenic stress by improving its antioxidant capacity, mainly through increasing the contents of antioxidant enzymes (POX, Peroxidases and CAT, Catalase), non-enzyme molecules and carotenoids[54]. However, some studies have shown that the detoxification effect of plasma on heavy metals in plants may be selective. After water spinach seeds or irrigation water were treated with plasma, the bioaccumulation factor (BCF) of Cd was significantly reduced, while the BCF of Pb did not change significantly, indicating that plasma treatment may inhibit the absorption of Cd without affecting the absorption of Pb[56].

2.5. Pathogen Infection

There are many harmful microorganisms in nature that can cause slow plant growth. APNTP treatment reduces the incidence and severity of Fusarium wilt of basil caused by Fusarium oxysporum f. sp. Basilici in sweet basil[63]. Although plasma-activated water did not show direct antimicrobial activity against Xanthomonas vesicatoria, which causes bacterial leaf spot of tomato, it enhanced the defense capacity of tomato plants, increasing the relative protection by 61% in vitro [60]. Low-pressure plasma treatments have demonstrated to be efficacious in the suppression of fungal crop diseases, including boil smut of maize, root rot of lupine and winter wheat[61]. This is due to reduced levels of seed infection, stimulation of germination in the field, early seedling growth, and increased resistance to pathogens during plant growth[61]. Plants treated with plasma also showed delayed disease progression and reduced disease severity. Tomatoes treated with APNTP showed a significant improvement of resistance to bacterial wilt caused by Ralstonia solanacearum [25]. Studies have shown that plasma treatment can lead to a significant reduction in fungal load, with untreated seeds exhibiting a pathogenicity rate of 58%, while the seeds treated with plasma demonstrated pathogenicity rates of 6.7% and 13.3%. Vaccination effects of plasma were also demonstrated against bakanae disease caused by the fungal pathogen Fusarium fujikuroi and bacterial seedling rot caused by Burkholderia glumae [59]. APNTP can also be used as a method of physical disinfection for plant seeds. Microbial contamination can lead to reduced seed viability, low germination rates, and delayed germination. The study found that after two minutes of corona discharge plasma jet treatment, the number of microorganisms on the seeds, primarily aerobic bacteria, molds, yeasts was significantly reduced (up to 99%)[30].

Recent mechanistic studies have elucidated that plasma treatment confers multi-stress tolerance through diverse molecular and biochemical mechanisms. At the epigenetic level, plasma induces DNA demethylation of abscisic acid catabolism genes and α-amylase (starch-hydrolyzing enzyme) genes, thereby reversing heat stress-induced germination inhibition[51]. Under low-temperature stress, plasma activates phosphotransferase activity, modulates phosphorus compound metabolism, and triggers defense responses that maintain cellular homeostasis[52]. Plasma exposure also significantly enhances the activities of catalase (CAT) and ascorbate peroxidase (APX), which scavenge excessive reactive oxygen species and confer drought stress tolerance[43]. Furthermore, non-thermal plasma treatment upregulates CAT and superoxide dismutase (SOD) activities while inducing stress-responsive genes (including chitinase and xylanase inhibitor protein genes), thereby enhancing seed tolerance to salt-alkali stress[46]. Notably, plasma exhibits dual functionality by simultaneously promoting plant stress tolerance and exerting antimicrobial effects. The antimicrobial properties are mediated through plasma-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS), including hydroxyl radicals (•OH), peroxyl radicals (•HO₂), superoxide anions (•O₂⁻), ozone (O₃), and singlet oxygen (¹O₂), nitric oxide (NO), nitrogen dioxide (NO₂), and nitrate (NO₃⁻), which disrupt microbial membrane integrity and cellular functions[65].

3. Plasma-Mediated Responses to Environmental Stresses in Animals

Currently, there are serious challenges to the habitats of animals and humans, such as pathogen infections, physical trauma, organic poisoning and air pollution[72] (Figure 3). These problems not only affect the health and productivity of animals but also threaten the sustainable development of society[73]. Similarly, many studies have found application prospects for plasma technology in animal fields, including sterilization, wound healing, pollutant degradation, animal breeding and air purification (Table 2 and Table 3). To elucidate the effect of plasma on animal resistance to environmental stress, we summarize and discuss the role and mechanisms of plasma technology in addressing the above-mentioned environmental and health challenges (Figure 3 and Figure 4).

3.1. Pathogen Infection

Contamination of frequently touched surfaces and equipment on farms can harm animal health, leading to reduced productivity and economic losses[105]. Microorganisms present in farms can cause mastitis and endometritis in cows, as well as gastrointestinal and respiratory diseases[106]. This also includes a broader range of diseases such as African swine fever, classical swine fever, porcine reproductive and respiratory syndrome, rinderpest, foot-and-mouth disease, and bluetongue disease, among others[107].Therefore, sterilization and disinfection of animal farms facilitate a pathogen-free environment essential for the growth and development of animals[90,108]. APNTP effectively inactivates microbial pathogens including Staphylococcus aureus, E. coli, vancomycin-resistant Enterococci, Acinetobacter baumannii, and herpes simplex virus type 1 (HSV-1)[90,95,109]. In addition, APNTP is an effective, non-toxic, and equipment-safe sterilization method that can sterilize small items such as cages, feeding utensils, and drinking water equipment in animal farms and also effectively disinfects large surfaces such as walls, floors, and fences[93]. Studies have shown that APNTP disinfection has little effect on the quality of metals such as stainless steel, copper, tin-copper, and passive electronic components, and they can all be used normally after this process[93]. Another study demonstrated that after treatment with plasma, the cell walls of Gram-negative bacteria (GNB), including P. aeruginosa, E. coli, and Vibrio parahaemolyticus, exhibited irreversible perforations, leading to the release of intracellular compounds such as proteins and DNA. In contrast, for Gram-positive bacteria (GPB), including Listeria monocytogenes and S. aureus, intracellular components underwent oxidation and release[91]. It has been reported that APNTP disinfection technology has become an ideal choice for animal farms due to its high efficiency, lack of residue, environmental protection, and non-toxicity[92,110]. Plasma has been found to reduce bacterial burden in wounds and promote healing[84]. In conclusion, it can be demonstrated that plasma has the capacity to decontaminate wounds effectively and inactivate various pathogens, including viruses, bacteria, and fungi, thereby promoting wound healing[111,112].

Recent studies have found that plasma can be used to generate vaccine effects, indirectly improving the resistance of animals and humans to the environment[113,114]. Plasma can destroy the proteins and genome of viruses by producing active particles (including ROS, RNS and UV photons), thereby causing them lose their pathogenicity[93,94]. It has been reported that Newcastle disease virus (NDV, LaSota) strains and H9N2 avian influenza virus (AIV, A/Chicken/Hebei/WD/98) can be completely inactivated by APNTP treatment for two minutes, and NDV vaccine can induce high titer NDV-specific antibodies in specific pathogen free (SPF) chickens by APNTP treatment for two minutes[115]. In summary, APNTP, as an emerging inactivating agent, has the advantages of high efficiency, safety, and stability. Vaccines treated with APNTP have obvious advantages in immunogenicity and protective effects and have broad application prospects.

3.2. Physical Trauma

Helium/argon cold atmospheric plasma jet (He/Ar-CAPJ) treatment was found to accelerate wound healing by promoting granulation tissue formation and reducing inflammation in skin tissue in a study on wound healing in rats[77]. In addition, N₂-generated non-thermal plasma promotes wound repair by inducing epithelial-mesenchymal transition (EMT) and activating the matrix metalloproteinase-9/urokinase-type plasminogen activator (MMP-9/uPA) system in a scratch wound healing assay[74]. Similar results were obtained when investigating the therapeutic effects of a non-thermal microplasma jet device array on second-degree burns in a rat model, where plasma reduced the trauma area and was able to modulate inflammatory gene expression, again contributing to epithelial tissue regeneration[116]. Studies showed that APNTP promoted neuron regeneration in rats[82]. In the study of the effects of APNTP on functional recovery after sciatic nerve damage (ND), it was found that APNTP treatment could restore the level of the sciatic nerve by 60%[76]. In addition, after seven days following the treatment of Sprague–Dawley (SD) rats with muscle defects using APNTP, a significant increase in the number of satellite cells was observed compared to the untreated group, demonstrating that APNTP also has a beneficial effect on muscle repair after injury[81].

Plasma is also considered a novel approach for fracture treatment. White rabbits showed ideal bone regeneration after 10 minutes of APNTP treatment, and that the volume of new bone in the plasma-treated group was 1.51-fold greater than in the untreated group[81]. Plasma has also been demonstrated to inhibit scar formation following injury. Plasma jet treatment of injured mice demonstrated that the scar area of plasma-treated rats was observably smaller than that of the plasma-untreated group at 21 postoperative days, and the degree of epithelialization of the scars was significantly higher than that of the untreated group[78]. In addition, plasma treatment of rat wounds accelerates wound healing, reduces scar area and inhibits the formation of scar signaling pathways such as TGF-β1, p-Smad2 and p-Smad3[78,80]. Overall, plasma has a beneficial effect on wound healing, tissue regeneration, cell proliferation, cell migration and angiogenesis.

3.3. Insufficient Fertility and Reproduction

APNTP technology can be used as a new technology to help solve some problems in animal fertility. Current studies have shown that APNTP technology can be used to promote the growth of rooster embryos[86]. The sperm quality-promoting effect of plasma treatment was regulated by the significant improvements of adenosine triphosphate production and testosterone level, and by the modulation of reactive oxygen species balance and adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathway in the spermatozoa[89]. According to previous study, APNTP affects the fertility of animals by mediating ROS balance, energy metabolism and antibacterial effects[87,117]. APNTP can induce intracellular ROS production, and the balance between ROS production and clearance activity is necessary to ensure optimal sperm quality. A certain amount of ROS can stimulate sperm energy acquisition, the acrosome reaction, and chemotactic and oocyte fusion. The effect of plasma may be dose-dependent, and plasma can promote growth and development under appropriate conditions.

3.4. Organic and Inorganic Poisoning (Pesticide Residues, Antibiotics, Mycotoxins)

There is a wide variety of pollutants in the environment, mainly in water, food and soil, including pesticide residues, antibiotics, microplastics and mycotoxins, among others. Most of these pollutants have a variety of toxic effects such as organ toxicity, neurotoxicity, developmental and reproductive toxicity, immunotoxicity, etc., and pose serious threats to animal health. For example, patulin is a neurotoxic secondary metabolite with strong toxicity to animal tissues and cells, and has been reported to cause renal congestion, pulmonary edema, peritoneal and pleural effusion, and tissue necrosis in mice[97]. Chemical residues (e.g., organic dyes, fertilizers, and pesticides) that can be toxic, mutagenic, and carcinogenic to humans, have been linked to a wide range of diseases, such as cancers, hormonal disorders, asthma, allergies, reproductive disorders, neurological disorders, skin disorders, visual disturbances, and immune system-related disorders. APNTP has received much attention in the detoxification and removal of pollutants in water[97,98]. It has been demonstrated that APNTP can effectively degrade pesticide residues such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) by destroying the chemical structure of organic pollutants through the oxidation by ROS and reactive nitrogen species (RNS)[96]. In plasma reactions, introduction of ambient air into an argon-air plasma jet introduces an abundant source of ROS and RNS, such as hydroxyl radicals (OH), monoline oxygen (O2*), nitric oxide (NO), nitrate (NO₃), nitrogen trioxide (N₂O₃), and nitrogen pentoxide (N₂O₅). Therefore, argon-air plasma injection emerged as a promising solution for 4-NP degradation in wastewater with higher ROS and RNS generation without the need for a catalyst[99]. Low-temperature plasma treatment can significantly reduce pesticide (dichlorvos, malathion and endosulfan) content in water[118]. These degradation reactions are mainly caused by ROS and RNS in the plasma.

3.5. Air Pollution

Currently, with the rapid development of industries, chemical fuels and various air pollutants are being emitted into the air in large quantities, and this has led to an increase in airborne particulate matter. This increase in airborne particulate matter can lead to a range of respiratory diseases in humans and animals, such as asthma, lung cancer, acute respiratory infections, and chronic bronchitis[119]. Many types of particulate matter can be easily inhaled and adhered to the respiratory tract, trachea, bronchus and even alveoli. The products of the reaction of NO and SO₂ with the generated aerosol particles of NaClO₂, NO₂ gas, and aqueous ions such as NO^2-, NO^3-, HSO^3-, and SO₄^2- lead to inflammation, oxidative stress, and fibrosis[120]. Studies have found that plasma technology not only has the potential to improve air quality and reduce air pollution but also has the advantage of being more energy efficient than other technologies[102]. In the food industry, plasma has been reported to improve air quality and eliminate volatile organic compounds. The high-energy electron bombardment in APNTP can change ground-state molecules to their sub-stable or excited states, and the products undergo multi-level physical and chemical reactions to form ions and free radicals, which react with gaseous pollutants and intermediates produced by electron collisions with precursors to ultimately produce harmless products[102]. APNTP can also break down formaldehyde (HCHO), a typical volatile organic compound in indoor air, and convert it to CO₂ and H₂O[103]. Moreover, APNTP is more energy efficient than other technologies and can remove multiple air pollutants simultaneously[121].

In summary, accumulating evidence demonstrates that plasma accelerates wound healing through coordinated regulation of multiple molecular mediators. Plasma modulates oxidative stress mediators (iNOS and NO), pyroptotic mediators (NOD-like receptor protein 3, Caspase-1, and Interleukin-1β), and angiogenesis mediators (vascular endothelial growth factor and angiopoietin-1)[122]. The wound healing process involves activation of critical signaling pathways, including yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ), β-catenin, and TGF-β[123]. These molecular cascades accelerate lipid metabolism, facilitate fibrin deposition, and enhance granulation tissue formation, neovascularization, and re-epithelialization[124]. Furthermore, plasma exhibits potent antimicrobial properties by significantly reducing wound bacterial burden. This antimicrobial effect is mediated through Rac-GTP-dependent activation of NADPH oxidase 2 (NOX2), which generates superoxide anions that eliminate pathogens[125]. In the context of fertility and reproduction challenges, plasma treatment significantly improves sperm quality through multiple mechanisms. Plasma-treated sperm display increased progressive motility and viability, decreased reactive oxygen species (ROS) levels, and upregulated expression of key proteins including kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (NRF2), NAD(P)H quinone dehydrogenase 1 (NQO1) and glutathione peroxidase 8 (GPx8)[126]. Mechanistically, DBD plasma modulates sperm ROS homeostasis through miRNA-mediated regulation of the KEAP1/NRF2 signaling pathway[126]. Furthermore, DBD plasma increases exon methylation levels of the AMP-activated protein kinase (AMPK) gene, thereby improving glycolytic flux, mitochondrial function, and antioxidant capacity. Importantly, these beneficial effects are achieved without compromising sperm DNA integrity or acrosome structure, ensuring reproductive viability[117].

Plasma-mediated degradation of organic and inorganic pollutants is primarily based on advanced oxidation processes (AOPs). Plasma-generated reactive oxygen and nitrogen species (RONS) ultimately reach water or gas phases containing antimicrobial agents and organic contaminants. RONS with the highest oxidation potentials preferentially attack pollutants, triggering oxidation, mineralization, and molecular backbone fragmentation or addition-elimination reactions at specific functional groups or atoms[127,128]. Beyond RONS, plasma generates solvated electrons, ultraviolet radiation, thermal energy, and electromagnetic fields. These synergistic factors trigger cascade reactions that continuously degrade organic compounds, ultimately achieving degradation and inactivation of multiple hazardous substances in liquid or solid matrices[128].

4. Regulatory Mechanisms of Plasma-Mediated Promotion of Tolerance to Environmental Stresses

4.1. Plasma Activated Signaling Pathways

The transforming growth factor-β (TGF-β1)/ small mothers against decapentaplegic (Smad)2/3 pathway is one of the important signaling pathways in scar formation. Following plasma treatment, the TGF-β/Smad2/3 pathway upregulates extracellular matrix remodeling genes, including collagen type I (COL1A1), fibronectin, and α-smooth muscle actin (α-SMA), all of which orchestrate tissue regeneration and wound healing processes[78]. Vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 (VEGFA/VEGFR2) is an angiogenesis-related signaling pathway. Cold atmospheric pressure plasma (CAPP) induces phosphorylation and activation of endothelial NO synthase (eNOS). This activity increases the level of endogenous NO in endothelial cells, which in turn stimulates VEGFA/VEGFR2 signaling to regulate angiogenesis in vitro[80]. Wingless/Int (Wnt) is a family of signaling proteins involved in various developmental functions. Expression levels of Wnt-related genes that promote cell proliferation in the neural tube after NTP treatment were significantly increased, confirming that NTP-treated retinoic acid (RA) differentiated SH-SY5Y (human neuroblastoma cells) cells can stimulate Wnt signaling to promote nerve regeneration[82]. The (phosphatidylinositol 3-Kinase / protein kinase B / mammalian target of rapamycin) PI3K/AKT/mTOR signaling pathway is critical for cell proliferation. Treating HaCaT cells with low doses of NTP was found to promote cell proliferation by activating the PI3K/AKT/mTOR signaling pathway, and the increased expression of signal transducer and activator of transcription 3 (STAT3) and Cyclin D1 was observed[129]. Non-thermal plasma treated solution (NTS) acts on BEAS-2B (human bronchial epithelial cell line cells similar to nasal mucosal epithelium) and is found to stimulate epithelial-mesenchymal transition (EMT) signaling pathways to cell migration to promote skin wound healing[76].

In plants, atmospheric pressure-cold plasma (APCP) treatment of early Arabidopsis seedlings can regulate the expression of adversity-responsive genes through the mitogen-activated protein kinase (MAPK) signal transduction pathway, thereby promoting Arabidopsis seedling growth[62]. Plasma can also downregulate the expression of viral genes angiotensin-converting enzyme 2 (ACE-2), spike (S) gene, and RNA-dependent RNA polymerase (Rd/Rp) helicase gene, as well as the protein expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ), cyclooxygenase-2 (COX-2), C-X-C motif chemokine ligand 10 (CXCL-10), and C-C motif chemokine ligand 5 (CCL-5) through the NF-κB and MAPK pathways[130]. H₂O₂ and abscisic acid (ABA) are signaling pathways associated with cold stress resistance. The application of cold plasma (CP) as a seed treatment has been demonstrated to induce the upregulation of H₂O₂ and ABA cascade signaling, which in turn upregulates the cold-acclimated inducer of c-repeat binding factor expression 1(ICE1) and c-repeat binding factor (CBF) genes, thereby enhancing the cold resistance of tomato plants[50]. Elevated levels of H₂O₂ and NO present in cold atmospheric plasma activation have the capacity to induce the MAPK signaling pathway, thereby stimulating the expression of pathogenesis-related (PR) proteins, antioxidant enzyme activity, and defense hormone (salicylic acid and jasmonic acid) pathway enzymes. When Arabidopsis plants infected with cucumber mosaic virus strain yellow (CMV(Y)) were exposed to nitrogen pentoxide (N₂O₅) generated by plasma treatment, the resistance of Arabidopsis to CMV was enhanced, and N₂O₅ could enhance Arabidopsis immune function by relying on the ethylene (ET) signaling pathway[64].

4.2. Plasma Activation of Related Genes in Response to Environmental Stress

APNTP can make cells exhibit unusual physiological states by activating important regulatory genes in cells[131]. This characteristic provides a new direction for APNTP-mediated resistance of external environmental stimuli[132]. Low-temperature cold plasma treatment of wounds inhibits generation of proinflammatory factors such as TNF-α (Tumor Necrosis Factor-alpha), IL-6 (Interleukin-6), IL-1β (Interleukin-1 beta), and promotes the expression of VEGF (Vascular Endothelial Growth Factor), bFGF (Basic Fibroblast Growth Factor), and TGF-β (Transforming Growth Factor-beta) in tissue[85]. After fibroblasts are treated with low temperature plasma, P56 can be phosphorylated and the expression of proteins such as P50 can be activated to promote the recovery of fibroblasts at the wound[75]. In animals, APNTP treatment under appropriate conditions can promote the expression of growth-related factors such as thyroid hormones and growth hormones, and promote the growth efficiency of animals[32]. Some products of photosynthesis also play a key role in plant immunomodulation[133]. In terms of genes regarding plant dehydration resistance, it was found that the expression of SnRK2 related genes of ABA (abscisic acid) was upregulated, thereby improving the drought resistance of plants[40].

4.3. Plasma-Enhanced Antioxidant Defense System

Antioxidant enzymes are organic molecules that maintain cellular redox balance in organisms. Treatment of wounds in diabetic patients with APNTP showed a significant decrease in protein expression of superoxide dismutase (SOD), reducing oxidative stress levels[80]. Chicken sperm obtained low levels of ROS and malondialdehyde (MDA) after exposure to APNTP, and the increased activity of SOD, Catalase (CAT), and Glutathione Peroxidase (GPx) enhanced antioxidant defense improving chicken sperm mobility[87]. The same study showed that the concentration of SOD, CAT, and GPx in chicken serum after plasma treatment significantly increased, which promoted the growth and development of chickens[32].

Similar results were obtained in plants after plasma treatment. Signal molecules produced by plasma, especially ozone, nitric oxide and/or ultraviolet radiation, induced an increase in peroxidase and phenylalanine ammonia lyase (PAL) activities[134]. DBD plasma treatment significantly improves the SOD, catalase (POD) and CAT activities of wheat seedlings under drought stress, accelerating the removal of ROS, thereby reducing oxidative damage and helping to maintain normal physiological metabolic activity[40]. Similar studies found that low-temperature plasma treatment enhances the SOD and CAT activities of rapeseed seedlings under drought stress and improves their growth[39].

Ar/Air plasma treatment helped detoxifying cadmium (Cd) in wheat, where plasma alleviated Cd-induced H₂O₂ rise and oxidative damage in tissues by inducing upregulation of antioxidant enzymes (SOD and CAT) and their corresponding genes (TaSOD and TaCAT)[53]. Cold plasma reduces bacterial blight symptoms caused by R. solanacearum in tomatoes by enhancing the activity of POD, polyphenol oxidase (PPO) and PAL[25]. In summary, plasma treatment can improve wound healing and development in animals by reducing the oxidative stress level and alleviate hindered growth and development in plants caused by oxidative stress.

4.4. Plasma Reduces the Threat of Harmful Microorganisms to Plants and Animals

Studies have shown that plasma therapy can effectively inhibit the activity and pathogenicity of microorganisms. The use of cold atmospheric surface microdischarge plasma devices can effectively inactivate bacteria and yeast pathogens in the air[135]. Various treatments using non-thermal plasma can lead to morphological changes in the mycelium structure of plant pathogenic fungi (F. oxysporum, B. cinerea, and A. alternata) and severely reduce pathogenicity[136]. ROS, RNS and acid produced by plasma can completely react with polysaccharides, lipids, proteins and nucleic acid molecules in biofilms, thereby destroying the cell membrane and inactivating microorganisms[137,138]. Furthermore, studies have shown that UV light in plasma affects the upper layer of the biofilm matrix, allowing ROS and RNS to enter. It can also destroy protein content in the outer membrane by weakening chemical bonds[138]. In a similar manner, hydrogen peroxide, produced in plasma activated water (PAW), can undergo a secondary reaction with iron substances and the downstream products of this reaction cause DNA damage, which in turn can lead to mutations in biofilm-producing cells[139].

In addition, ROS and RNS generated by plasma can directly interact with specific intracellular molecules, thereby participating in a variety of physiological processes. In plant cells, H₂O₂ function as signaling molecules that activate calcium channels in guard cells, triggering stomatal closure under drought stress. This H₂O₂-mediated calcium influx represents a critical early response in ABA-dependent stress signaling pathways[140]. plasma-derived NO acts as a mobile signaling molecule that can diffuse across membranes and modify cysteine residues in target proteins through S-nitrosylation, thereby modulating protein function and stress response pathways[141]. In bacterial cells, ROS and RNS cause oxidative damage to membrane phospholipids through lipid peroxidation, resulting in increased membrane permeability and leakage of intracellular contents. For Gram-negative bacteria, this oxidative attack creates irreversible perforations in the cell wall, leading to release of proteins and DNA, while in Gram-positive bacteria, the reactive species penetrate the thicker peptidoglycan layer to oxidize intracellular components directly[138]. ROS also interact with nucleic acids by inducing strand breaks and base modifications in DNA[142]. In mammalian wound healing, plasma-generated ROS stimulate the phosphorylation of endothelial nitric oxide synthase (eNOS), which increases endogenous NO production. These ROS also enhance the expression of pro-angiogenic factors including platelet-derived growth factor receptor (PDGFR) and cluster of differentiation (CD31). Together, these effects activate the VEGFA/VEGFR2 signaling pathway to promote angiogenesis[143]. The dose-dependent nature of these interactions is critical, as moderate ROS levels activate protective antioxidant enzyme systems (SOD, CAT, POD) and stress-responsive genes, while excessive ROS accumulation leads to cellular damage through protein carbonylation and membrane disruption[144].

5. Limitations, Challenges, and Adverse Effects of Plasma Applications

While APNTP technology shows considerable promise, several limitations and potential adverse effects must be acknowledged. First, excessive ROS and RNS production can lead to oxidative damage, including lipid peroxidation, protein denaturation, and DNA strand breaks, particularly when treatment parameters are not optimized. Studies have reported that prolonged or high-intensity plasma exposure may inhibit seed germination, reduce cell viability, and even induce mutagenic effects in certain plant and animal models[145]. Second, the biological responses to APNTP are highly dose-dependent and species-specific, resulting in significant variability and occasional contradictory findings across experiments[146].

In addition, A critical barrier to widespread adoption and clinical/agricultural translation of APNTP technology is the lack of standardization in treatment protocols, which severely limits reproducibility, comparability, and establishment of universal guidelines[147]. Different plasma generation devices produce dramatically different outputs. Dielectric barrier discharge, plasma jets, corona discharge, and microwave plasma systems generate distinct profiles of reactive species, electron densities, gas temperatures, and spatial distributions[148]. Even when targeting the same biological tissue under seemingly identical conditions, device-specific characteristics can lead to fundamentally different biological outcomes (Table 4). And the choice of working gas fundamentally determines plasma chemistry and biological effects. Inert gases like argon and helium generate high concentrations of metastable excited particles and UV photons, nitrogen-containing gases such as air and N₂ produce abundant reactive nitrogen species (RNS: NO, NO₂, N₂O₅, ONOO⁻)[149,150,151]. Moreover, treatment parameters including voltage, frequency, pulse width, duty cycle, exposure time, target distance, and gas flow rate collectively determine the effective "dose". These dose-response relationships are highly species-specific and context-dependent[150]. Finally, Treatment modality further determines biological outcomes. Direct plasma exposure delivers a complex mixture of reactive species, UV radiation, electric fields, and charged particles in a single step, resulting in rapid and potent biological effects. By contrast, indirect methods using plasma-activated water or treated gases depend on long-lived chemical species (H₂O₂, NO₂⁻, NO₃⁻) that persist in the medium and act over extended timeframes[151]. To advance practical implementation of plasma technology, we advocate for the adoption of unified dosimetry metrics including energy dose per unit area (J/cm⁻²), concentrations of key reactive species (H₂O₂, NO₂⁻, O₃), and UV flux measurements. Systematic dose-response studies should be conducted for each specific application scenario, target organism, and stress type. We also recommend establishing open-access databases documenting plasma treatment parameters alongside biological outcomes. These efforts will facilitate standardization and accelerate the translation of APNTP technology from laboratory to field applications.

6. Conclusions and Future Perspectives

As an emerging technology, APNTP has shown wide application prospects in agriculture and animal health. Research shows that APNTP has a significant promoting effect on plant growth, stress resistance and animal health maintenance, and its action mechanisms show certain commonalities in animals and plants. In the field of agriculture, plasma treatment can significantly increase plant resistance to extreme weather, soil pollution and biological stress. Plasma can promote plant vegetative growth, root development and seed germination by activating specific signaling pathways and gene expression. In addition, plasma-treated plants exhibit stronger antioxidant capacity, primarily by enhancing the expression of antioxidant enzymes. APNTP also shows many advantages in animal health. It can effectively eliminate microorganisms in the animal feeding environment, reduce the use of chemical reagents, remove environmental pollution, promote wound healing and reduce animal deaths caused by accidental trauma. APNTP treatment also reduces the ammonia produced during animal feeding and the risk of respiratory diseases in animals and improve animal welfare. It should also be noted that plasma can increase animal sperm activity, which is of great significance in dealing with the problem of declining animal fertility. As a technology with wide application prospects, APNTP has great potential in fighting environmental stress. This review provides a theoretical basis for the future application of plasma technology in different fields.

Despite these promising results, several critical challenges must be addressed before APNTP can transition from laboratory research to widespread field applications. The foremost challenge is the lack of standardization in treatment parameters across studies. Variations in plasma source types, gas composition, treatment duration, and energy input hinder the establishment of reproducible protocols and complicate cross-study comparisons. In addition, The biological effects of plasma are fundamentally mediated by reactive oxygen and nitrogen species, which operate within a narrow therapeutic window. Excessive RONS production can induce oxidative stress beyond cellular antioxidant capacity, resulting in lipid peroxidation, protein carbonylation, and damage to nucleic acids. Several studies have reported DNA strand breaks and chromosomal aberrations following high-intensity or prolonged plasma exposure, raising concerns about potential mutagenic effects. In agricultural applications, unintended genetic alterations in crop germplasm could compromise breeding programs and food safety. In animal health contexts, germline mutations could have transgenerational consequences. Moreover, the scarcity of cost-benefit analyses in current literature makes it challenging to evaluate the economic competitiveness of plasma technology compared to conventional approaches. Although plasma treatment can reduce pesticide dependence, whether the associated equipment costs and energy consumption outweigh the savings from decreased chemical inputs remains uncertain, especially in small-scale agricultural contexts.

From a mechanistic perspective, future investigations should employ multi-omics approaches integrating transcriptomics, proteomics, and metabolomics to elucidate the complex molecular networks underlying plasma-induced stress tolerance. Such comprehensive analyses will provide deeper insights into the coordinated cellular responses activated by APNTP treatment. Furthermore, integrating APNTP with complementary sustainable technologies, such as precision agriculture, biochar amendments, or beneficial microorganism inoculation, may yield synergistic effects that enhance overall system resilience. Establishing international collaborative networks and open-access databases documenting standardized treatment protocols and outcomes would accelerate knowledge translation and facilitate evidence-based implementation strategies across diverse agricultural and animal production systems.

Contributions: Conceptualization, N-N. Y. and GP.; formal analysis, N-N. Y. and SJ; investigation, W-MS. and WK.; writing—original draft preparation, N-N. Y. WK. and GP.; writing—review and editing, N-N. Y. W-MS. SJ. and GP; supervision, H-NS. and GP; project administration, GP.; funding acquisition, GP.