Antioxidant Defense System in Plants: Reactive Oxygen Species Production, Signaling, and Scavenging During Abiotic Stress-Induced Oxidative Damage

Abstract

Plants face various abiotic stresses in their natural environments that trigger the production of reactive oxygen species (ROS), leading to oxidative stress and potential cellular damage. This comprehensive review examines the interplay between plant antioxidant defense systems and ROS under abiotic stress conditions. We discuss the major enzymatic antioxidants, including superoxide dismutase, catalase, reductases, and peroxidases, as well as non-enzymatic antioxidants, such as ascorbic acid, glutathione, polyphenols, and flavonoids, which play crucial roles in ROS detoxification. This review elaborates on different types of ROS, their production sites within plant cells, and their dual role as both damaging oxidants and key signaling molecules. We discuss how various abiotic stresses—including heat, cold, drought, flooding, salinity, and heavy metal toxicity—induce oxidative stress and trigger specific antioxidant responses in plants. Additionally, the mechanisms of ROS generation under these abiotic stress conditions and the corresponding activation of enzymatic and non-enzymatic scavenging systems are discussed in detail. This review also discusses recent advances in understanding ROS signaling networks and their integration with other stress-response pathways. This knowledge provides valuable insights into plant stress-tolerance mechanisms and suggests potential strategies for developing stress-resistant crops by enhancing antioxidant defense systems. Moreover, the strategic ROS modulation through priming, exogenous antioxidants, nanoparticles, or genetic tools can enhance plant resilience. Integrating these methods with agronomic practices (e.g., irrigation management) offers a sustainable path to climate-smart agriculture. Our review reveals that ROS accumulation can be detrimental; however, the coordinated action of various antioxidant systems helps plants maintain redox homeostasis and adapt to environmental stress.

1. Introduction

Environmental stresses have emerged as significant threats to global agricultural productivity and sustainability [1]. The severity of climate change has a significant effect on plants’ ecosystems, with an increasing susceptibility of plants to multiple abiotic stresses, which influences their survival and productivity [1,2,3]. Abiotic stress includes temperature stress (heat and cold), drought, flooding, soil salinity, nutrient deficiency, heavy metal toxicity, and xenobiotic compounds [4,5,6]. These stresses are collectively responsible for approximately a 50% or more reduction in crop production [4,7,8]. Under field conditions, the combined abiotic stresses result in complex and synergistic effects on plant physiology, development, and metabolic processes, posing significant challenges for maintaining agricultural productivity and food security [1].

Plants have endogenous defense mechanisms that include both enzymatic and non-enzymatic antioxidant systems [9,10]. The enzymatic components consist of multiple specialized enzymes, including superoxide dismutase, catalase, various peroxidases, and reductases. Additionally, protective enzymes such as glutathione S-transferase, ferritin, polyphenol oxidase, and alternative oxidase play crucial roles during stress [10]. The non-enzymatic antioxidant molecules further strengthen the endogenous defense. These molecules include ascorbic acid, amino acids, phenolic acids, flavonoids, anthocyanins, carotenoids, vitamin E, and mineral elements [11,12,13,14,15,16]. These molecules are responsible for maintaining cellular redox homeostasis. Under normal conditions, plants maintain an optimal equilibrium between reactive oxygen species generation and neutralization. This equilibrium is essential for cellular signaling and regulating fundamental physiological processes, including plant growth and development [17,18]. However, the unfavorable environmental situation can disrupt this equilibrium by the excessive production of ROS, which leads to cellular damage and programmed cell death [18,19].

ROS serve a dual function in plant systems—while potentially harmful during stress (due to the overproduction of ROS that leads to oxidative stress), they also act as crucial signaling molecules that transmit stress signals to the nucleus via redox-mediated mitogen-activated protein kinase (MAPK) cascades. ROS can oxidize specific redox-sensitive proteins, initiating the MAPK cascade, and the activated MAPK can then phosphorylate transcription factors or other regulatory proteins, leading to changes in gene expression that help the plant adapt to stress [17,18,19]. These ROS molecules are involved in plant acclimation to environmental stress and function as key signal transducers that interact with several pathways [19]. ROS signaling is essential for fundamental biological processes such as cell division and cell differentiation [18]. Hydrogen peroxide (H₂O₂), a type of ROS, plays a central role in stress response across crop species, including cereals (rice, wheat, maize), legumes (mung bean, soybean), vegetables (cucumber), fruits (sour orange and strawberry), and herbs (basil) [20,21,22,23,24,25]. Recent studies showed complex crosstalk between ROS and other reactive species, such as reactive nitrogen species (RNS), reactive carbonyl species (RCS), and reactive sulfur species (RSS), in mediating plant responses to abiotic stresses [26,27,28]. This complex signaling network between ROS and other reactive species signifies a promising area of ongoing research in plant stress biology.

Our review examines the complex interplay of the enzymatic and non-enzymatic antioxidant defense of plants under various abiotic stresses. We highlight the types, production sites, ROS signaling, and their regulation in different stress conditions. In addition, we discuss the oxidative stress induced by different abiotic factors such as heat, chilling or cold stress, water scarcity, flooding, salinity, and heavy metal toxicity. By understanding these mechanisms, this review provides valuable insights for developing stress-tolerant crops and enhancing agricultural productivity in challenging environmental conditions.

2. Antioxidative Defense Mechanisms in Plants

Antioxidant defense mechanisms enable plants to cope with oxidative stress and maintain the redox balance during normal or unfavorable environmental conditions. Under abiotic stress, plants can upregulate their antioxidative machinery by activating the gene expression and post-translational modifications of key enzymes. This adaptive response enables plants to reduce oxidative damage at cellular levels (such as proteins, lipids, membranes, and nucleic acids) and maintain normal growth and development [29,30,31,32]. The enzymatic and non-enzymatic antioxidant defense system in plants operates in different cellular organelles. Enzymatic antioxidants directly neutralize reactive oxygen species or repair oxidative damage. Non-enzymatic antioxidants, including carotenoids, polyphenols, amino acids, vitamins, and minerals, also scavenge ROS and support enzymatic antioxidant activities to maintain the redox balance [1,2,33,34,35]. The antioxidant defense system is regulated by complex signaling networks involving various transcription factors, phytohormones, and secondary messengers that work collectively to maintain redox homeostasis at the cellular level.

2.1. Enzymatic Antioxidants

Enzymatic antioxidants directly quench the ROS or convert them to be the least reactive in order to protect cellular organelles during oxidative stress [9] (Figure 1). These enzymatic antioxidants include several enzymes such as superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), guaiacol peroxidase (GPx), glutathione S-transferase (GST), alternative oxidase (AOX), polyphenol oxidase (PPO), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), thioredoxin (Trx) including its multiple isoforms (f, m, h, s, o, x, y, and z), peroxiredoxin (PRX), and glutaredoxin (GRX) [9,10,33,34,35] (Figure 1). These enzymes can work independently or synergistically to maintain redox homeostasis (Table 1).

Superoxide dismutase (SOD) is the primary antioxidant defense enzyme that exists in three distinct metalloenzyme forms: Cu/Zn-SOD, Fe-SOD, and Mn-SOD [36]. These isoforms are in cytosol, chloroplasts, peroxisomes, and mitochondria [36,37]. The catalase, glutathione reductase, and ascorbate peroxidase work together to detoxify the H₂O₂ into water and oxygen molecules [38]. CAT and APX are metalloenzymes, which are located in peroxisomes and mitochondria [38]; however, APX is also found in cytosol, chloroplasts, microbodies, and in peroxisomal/glyoxysomal isoforms [39]. In chloroplasts, APX is a key component of the ascorbate–glutathione pathway (to produce monodehydroascorbate (MDHA)), requiring ascorbate as an electron donor [39]. This pathway, also known as the Foyer–Halliwell–Asada pathway, has both enzymatic (APX, DHAR, GPX, MDHAR) and non-enzymatic antioxidants (ascorbic acid and glutathione) that mainly regulate hydrogen peroxide levels in different cellular organelles [40]. The NADPH-dependent flavoprotein MDHAR produces ascorbate from MDHA by its thiol-mediated mechanism (Table 1). Spontaneous MDHA disproportionation yields dehydroascorbate (DHA), which is reduced to ascorbate via DHAR using glutathione as the electron donor. This process produces oxidized glutathione (GSSG), which is reduced to glutathione by NADPH-dependent glutathione reductase, thus maintaining redox equilibrium in the plant cells [41].

Glutathione reductase (GR) is a key enzyme that plays a vital role in cellular defense systems and exhibits the highest activity in chloroplasts (70–80%). However, GR is also present in other parts of the plant cell [42]. Several factors affect the function of GR, such as the environmental pH and availability of NADPH and oxidized glutathione. It maintains redox homeostasis during oxidative stress by catalyzing GSSG reduction using NADPH as the electron donor [42,43]. Meanwhile, GPX is another critical player in this system and a member of the thiol-containing peroxidase family; it neutralizes harmful ROS by using glutathione and thioredoxins (Table 1). GST facilitates the conjugation between glutathione (GSH) and various electrophilic compounds at its catalytic sites, enabling the detoxification of xenobiotics, particularly herbicides and pharmaceutical compounds, followed by their vacuolar sequestration [44]. Additional protective mechanisms include peroxiredoxins (Prx) and thioredoxins (Trx) [34,35]. Prx is a thiol-dependent enzyme that exhibits peroxidase-like functionality in cellular compartments such as cytosol, chloroplasts, mitochondria, and nucleus, whereas Trx proteins regulate cellular redox status through disulfide bond reduction [35]. Polyphenol oxidase (PPO) is localized in the thylakoid membranes of chloroplasts and exhibits direct modulatory effects on photosynthetic processes [45]. This enzyme demonstrates multifaceted antioxidant capabilities through its interactions with peroxidase systems and the water–water cycle [45]. These systems collectively form an intricate antioxidant network essential for cellular stress tolerance.

Table 1. Enzymatic antioxidants’ function during plant abiotic stress.

Serial No.	Enzymatic Antioxidant	Location	Function	Stress Response	Reference
1	Superoxide Dismutase (SOD)	Chloroplasts, mitochondria, cytosol	Catalyzes dismutation of O2•− to H2O2 and O2	Increases during drought, salinity, and heat stress	[9,46]
2	Catalase (CAT)	Peroxisomes	Converts H2O2 to H2O and O2	Upregulated during oxidative stress conditions	[47,48,49]
3	Guaiacol Peroxidase (GPx)	Cell wall, cytosol	Lignification Cell wall strengthening H2O2 scavenging	Response to mechanical stress and heavy metal stress	[9,50]
4	Ascorbate Peroxidase (APX)	Chloroplasts, cytosol	Reduces H2O2 to H2O using ascorbate as an electron donor	Enhanced activity during cold and drought stress	[9,40]
5	Glutathione Peroxidase (GPX)	Cytosol, chloroplasts	Reduces H2O2 and lipid hydroperoxides	Activated during heavy metal and UV stress	[9,40]
6	Glutathione Reductase (GR)	Chloroplasts, mitochondria	Maintains GSH/GSSG ratio by reducing GSSG to GSH	Elevated levels of salt and metal stress	[42]
7	Dehydroascorbate reductase (DHAR)	Chloroplasts, cytosol, mitochondria	Regenerates ascorbate from dehydroascorbate Maintains ascorbate pool	Enhances drought and salt tolerance; protects photosynthetic machinery	[42,51]
8	Monodehydro-ascorbate reductase (MDHAR)	Chloroplasts, cytosol, mitochondria, peroxisomes	Reduces mono-dehydroascorbate to ascorbate using NADPH Maintains cellular redox balance	Improves tolerance to oxidative stress Enhances aluminum stress tolerance	[9,52]
9	Peroxiredoxin (PRX)	Chloroplasts, mitochondria, nucleus	Reduces hydrogen peroxide and organic hydroperoxides	Scavenges peroxides and peroxynitrite (ONOO−); enhances heat drought, and cold tolerance	[34]
10	Thioredoxin (Trx)	Chloroplasts, mitochondria, cytosol	Maintains protein thiol–disulfide balance Maintains protein redox homeostasis and regulates enzyme activities	Protein disulfide reduction and redox regulation; improves drought and salt tolerance	[35]
11	Glutaredoxins (GRX)	Chloroplasts, mitochondria, nucleus, cytosol	Catalyzes thiol–disulfide exchange reaction Protein deglutathionylation	Glutathione-dependent protein repair, and iron–sulfur (Fe-S) assembly; enhances heavy metal tolerance	[53]
12	Glutathione S-transferase (GST)	Cytosol, chloroplasts	Conjugates glutathione to various substrates for detoxification	GSH conjugation and detoxification of lipid peroxidation products; improves drought and salt resistance	[54]
13	Alternative oxidase (AOX)	Mitochondrial inner membrane	Alternative respiratory pathway enzyme Reduces ROS production	Bypasses complexes III/IV in the mitochondrial electron transport chain; enhances drought and salinity stress tolerance	[55]
14	Polyphenol oxidase (PPO)	Chloroplasts	Oxidizes phenolic compounds into quinones	Oxidizes phenolics to quinones	[45].

Table 1. Enzymatic antioxidants’ function during plant abiotic stress.

Serial No.	Enzymatic Antioxidant	Location	Function	Stress Response	Reference
1	Superoxide Dismutase (SOD)	Chloroplasts, mitochondria, cytosol	Catalyzes dismutation of O2•− to H2O2 and O2	Increases during drought, salinity, and heat stress	[9,46]
2	Catalase (CAT)	Peroxisomes	Converts H2O2 to H2O and O2	Upregulated during oxidative stress conditions	[47,48,49]
3	Guaiacol Peroxidase (GPx)	Cell wall, cytosol	Lignification Cell wall strengthening H2O2 scavenging	Response to mechanical stress and heavy metal stress	[9,50]
4	Ascorbate Peroxidase (APX)	Chloroplasts, cytosol	Reduces H2O2 to H2O using ascorbate as an electron donor	Enhanced activity during cold and drought stress	[9,40]
5	Glutathione Peroxidase (GPX)	Cytosol, chloroplasts	Reduces H2O2 and lipid hydroperoxides	Activated during heavy metal and UV stress	[9,40]
6	Glutathione Reductase (GR)	Chloroplasts, mitochondria	Maintains GSH/GSSG ratio by reducing GSSG to GSH	Elevated levels of salt and metal stress	[42]
7	Dehydroascorbate reductase (DHAR)	Chloroplasts, cytosol, mitochondria	Regenerates ascorbate from dehydroascorbate Maintains ascorbate pool	Enhances drought and salt tolerance; protects photosynthetic machinery	[42,51]
8	Monodehydro-ascorbate reductase (MDHAR)	Chloroplasts, cytosol, mitochondria, peroxisomes	Reduces mono-dehydroascorbate to ascorbate using NADPH Maintains cellular redox balance	Improves tolerance to oxidative stress Enhances aluminum stress tolerance	[9,52]
9	Peroxiredoxin (PRX)	Chloroplasts, mitochondria, nucleus	Reduces hydrogen peroxide and organic hydroperoxides	Scavenges peroxides and peroxynitrite (ONOO−); enhances heat drought, and cold tolerance	[34]
10	Thioredoxin (Trx)	Chloroplasts, mitochondria, cytosol	Maintains protein thiol–disulfide balance Maintains protein redox homeostasis and regulates enzyme activities	Protein disulfide reduction and redox regulation; improves drought and salt tolerance	[35]
11	Glutaredoxins (GRX)	Chloroplasts, mitochondria, nucleus, cytosol	Catalyzes thiol–disulfide exchange reaction Protein deglutathionylation	Glutathione-dependent protein repair, and iron–sulfur (Fe-S) assembly; enhances heavy metal tolerance	[53]
12	Glutathione S-transferase (GST)	Cytosol, chloroplasts	Conjugates glutathione to various substrates for detoxification	GSH conjugation and detoxification of lipid peroxidation products; improves drought and salt resistance	[54]
13	Alternative oxidase (AOX)	Mitochondrial inner membrane	Alternative respiratory pathway enzyme Reduces ROS production	Bypasses complexes III/IV in the mitochondrial electron transport chain; enhances drought and salinity stress tolerance	[55]
14	Polyphenol oxidase (PPO)	Chloroplasts	Oxidizes phenolic compounds into quinones	Oxidizes phenolics to quinones	[45].

2.2. Non-Enzymatic Antioxidants

Non-enzymatic antioxidants play a crucial role in ROS detoxification through chain reaction interruption mechanisms [10]. Several key compounds, including ascorbic acid (AsA), glutathione (GSH), compatible solutes, phenolics, α-tocopherol, carotenoids, flavonoids, anthocyanins, and proline, work together to neutralize stress-induced ROS accumulation [56,57,58,59]. GSH, a vital thiol tripeptide, facilitates H₂O₂ degradation via glutathione peroxidase (GPX)-mediated reactions [13]. It functions within the AsA-GSH pathway as a reducing agent for dehydroascorbate reductase (DHAR) while also participating in H₂O₂ and lipid peroxide elimination through GPX and glutathione S-transferase (GST) conjugation reactions [13,60]. Ascorbic acid serves dual roles as an electron donor for ascorbate peroxidase (APX) and as a peroxidase cofactor [61]. Additionally, AsA supports tocopherol regeneration and xanthophyll synthesis, contributing to energy dissipation [62]. Carotenoids function as specialized light-harvesting pigments that mitigate oxidative stress under high light conditions through thermal energy dissipation [63]. These compounds also protect photosystem II by effectively quenching singlet chlorophyll, triplet chlorophyll, and singlet oxygen species in thylakoid membranes [63,64].

Osmoprotective compounds such as proline and glycine betaine significantly accumulate in plants during stress (

Table 2. Non-enzymatic antioxidants’ functions during plant abiotic stress.

Serial No.	Non-Enzymatic Antioxidant	Location	Function	Stress Tolerance	Reference
1	Ascorbic Acid (Vitamin C)	Chloroplasts, cytosol, vacuoles, mitochondria	Neutralizes ROS Regenerates vitamin E Acts as an enzyme cofactor in various physiological processes Key role in photoprotection	Acts as the first line of defense against ROS Protects photosynthetic machinery during heat/drought Maintains cell membrane integrity under salt stress Enhances cold tolerance Crucial for stomatal regulation during water stress	[61]
2	Glutathione (GSH)	Chloroplasts, mitochondria, cytosol, nucleus	Maintains cellular redox balance Detoxifies heavy metals Protects proteins from oxidation Regenerates ascorbate (AsA-GSH cycle)	Primary defense against heavy metal toxicity Maintains cellular redox state during oxidative stress Key role in xenobiotic detoxification Protects protein thiols during heat stress Essential for drought-tolerance mechanisms	[13]
3	Tocopherols (Vitamin E)	Cell membranes, chloroplast membranes, thylakoids	Protects membrane lipids Scavenges lipid peroxyl radicals (prevents lipid peroxidation) Maintains membrane stability Protects photosystem II	Prevents lipid peroxidation under stress Stabilizes membranes during temperature extremes Protects chloroplast function during high light stress Enhances drought resistance Critical for cold stress tolerance	[73]
4	Carotenoids	Chloroplasts, chromoplasts	Quenches singlet oxygen Protects chlorophyll Dissipates excess light energy	Protects photosynthetic apparatus from photo-oxidation Dissipates excess light energy as heat Stabilizes thylakoids under high temperatures Light stress adaptation by dissipating energy as heat	[63,64,74]
5	Phenolic Compounds	Vacuoles, cell walls, cytosol	ROS scavenging Metal chelation UV screening Cell wall lignification	Abiotic stress protection Enhanced pathogen resistance UV radiation screening Antioxidant activity	[75,76]
6	Flavonoids	Vacuoles, cell wall	UV-B radiation protection ROS scavenging Osmolyte function during drought Metal chelation	Strong UV-B radiation protection Drought stress tolerance Salt-stress amelioration Metal toxicity chelation	[58,77]
7	GABA (Gamma-aminobutyric acid)	Cytosol, mitochondria	Regulates stomatal closure Protects cellular membranes Maintains ion homeostasis Stabilizes membrane integrity Scavenges ROS Enhances antioxidant enzyme activities Maintains redox homeostasis	Chelates heavy metals Enhances frost tolerance Improves salt and drought tolerance Acts as a heat shock protectant Maintains photosynthetic efficiency under stress	[68,69,70]
8	Proline	Cytosol, chloroplasts	Osmolyte function Membrane stabilizer ROS scavenger Metal chelator Protein structure stabilizer	Osmolyte function during drought/salt stress Membrane stabilization under extreme temperatures ROS scavenging during oxidative stress Protein stabilization during dehydration Metal stress tolerance	[65]
9	Glycine Betaine	Chloroplasts, cytosol	Osmotic adjustment Protein structure protection Membrane integrity maintenance Photosynthetic apparatus protection ROS detoxification	Temperature stress tolerance Minimizes ROS during water deficit conditions. Cold stress protection	[66,67]
10	Selenium (Se)	Throughout plant tissues	Activates antioxidant defense mechanisms Enhances photosynthetic efficiency Enhances selenoproteins which combat ROS	Increases drought tolerance by regulating water status Protects against oxidative damage during salt stress Improves heavy metal stress tolerance	[78,79,80]
11	Zinc (Zn)	Cell wall, membranes, cytoplasm	Cofactor for antioxidant enzymes Maintains membrane stability Regulates stomatal function under drought Enhances root growth and water uptake	Regulates stomatal function during drought Involved in auxin metabolism and stress signaling Protects chloroplast structures during heat stress	[81]
12	Silicon (Si)	Cell walls, epidermis	Strengthens cell walls Improves water retention Forms a protective layer in cell walls Improves nutrients and water uptake	Improves water use efficiency during drought Reduces metal toxicity through complexation Enhances structural stability against lodging Regulates osmolyte accumulation under salt stress	[82,83]
13	Thiamine and nicotinic acid	Chloroplasts, mitochondria, and cytoplasm	Modulates MAPK and ABA pathways Upregulates phytochelatins Enhances GR, APX, and phenolic compounds Chelates lead (Pb) via phenolics/NADPH systems	Reduced Pb-induced oxidative damage (lower H2O2 and MDA) Enhanced activity of SOD, CAT, and POD, improving redox balance Higher photosynthetic efficiency	[84]

). The exogenous application of proline and glycine betaine represents a promising strategy for mitigating the detrimental impacts of environmental stresses on plants [65]. Glycine betaine (GB) has remarkable capabilities in enhancing plant tolerance to various abiotic stresses, especially high-temperature stress [66]. Extensive research has established GB as a crucial mediator of multiple physiological and molecular responses to heat stress, encompassing improvements in growth parameters, the protection of protein structures, the maintenance of photosynthetic efficiency, the modulation of stress-responsive gene expression, and the enhancement of antioxidant defense mechanisms [66,67]. Gamma-aminobutyric acid (GABA), an essential non-protein amino acid, demonstrates significant accumulation in plants during stress conditions, contributing to stress tolerance through its dual role in free radical scavenging and enzymatic regulation [68]. As an osmolyte, GABA either functions directly or stimulates the synthesis of other osmolytes like proline during drought conditions, facilitating osmotic adjustment for stress adaptation [69]. The GABA shunt pathway, comprising three key enzymes—glutamate dehydrogenase (GDH), GABA transaminase (GABA-T), and succinic semialdehyde dehydrogenase—mediates GABA metabolism [70]. Research on Arabidopsis thaliana has revealed that GABA-T gene deficiency results in decreased GABA and chlorophyll levels, impaired photosynthetic efficiency, and reduced GDH activity, while simultaneously increasing membrane ion leakage, malondialdehyde content, and accelerating leaf senescence under stress conditions [71]. Furthermore, GABA plays a crucial role in stress signal transduction through enhanced cytosolic calmodulin-dependent glutamate decarboxylase activity [72].

Recent advances in plant stress biology have highlighted the significance of vitamins and mineral elements in stress tolerance (Table 2). Some vitamins, such as thiamine (vitamin B₁) and nicotinic acid (vitamin B₃), enhance plant heavy metal stress tolerance by modulating antioxidant defense systems and redox homeostasis. Their exogenous application improves growth, reduces oxidative damage, and restricts Pb accumulation in Lens culinaris, offering a sustainable strategy for crop protection in contaminated soils [84]. Similarly, mineral elements such as silicon (Si) application have emerged as a promising approach for enhancing plant drought stress tolerance [82]. Additionally, the foliar application of zinc particles enhanced the antioxidative defense of sunflower plants against drought stress [81]. Similarly, selenium nanoparticles demonstrated superior bioactivity and safety profiles at low concentrations, exhibiting enhanced biocompatibility and reduced toxicity compared to conventional selenium forms like selenate and selenite [85]. The integration of these non-enzymatic antioxidant compounds in agricultural practices represents a sustainable approach to enhancing crop resilience against various abiotic stresses, ultimately contributing to global food security in the face of climate change.

3. Types, Production Sites, and Signaling of Reactive Oxygen Species

Reactive oxygen species exhibit dual functionality in plant cells based on their concentration levels [17]. At lower levels, they serve as vital signaling molecules, facilitating cellular responses to environmental stress through complex signaling cascades [17]. However, during severe abiotic stress, excessive ROS accumulation leads to cellular toxicity and triggers programmed cell death [86]. The cellular impact of ROS regulatory, harmful, or signal mediating is determined by the precise equilibrium between their generation and scavenging processes [19]. The endogenous antioxidative defense systems scavenge ROS and maintain cellular redox homeostasis [87,88,89]. To enhance plant stress tolerance, cellular antioxidant capacity can be improved by genetic modifications (transgenic plants) or foliar application, thereby fortifying the plant’s defense against environmental stress.

3.1. Types of ROS

Reactive oxygen species are a diverse group of molecules that include free radicals and non-radical compounds [90]. The free radicals include superoxide (O₂•⁻), alkoxyl (RO•), hydroxyl (•OH), and peroxyl (ROO•) radicals, whereas H₂O₂, singlet oxygen (¹O₂), and ozone (O₃) are considered non-radical molecules. The non-radical ROS molecules are less reactive but play important roles in plants. Beyond these, there are other non-radical ROS molecules such as organic hydroperoxides (ROOH), excited carbonyls (RO*), and hypochlorous acid (HOCl) [90,91].

Reactive oxygen intermediates (ROIs) are a specific subset of ROS that form when O₂ is partially reduced. This group includes O₃ and ¹O₂ molecules. The ROS family is quite extensive and further includes reactive species such as hypohalous acids (HOBr, HOI, HOCl) and radical forms like carbonate (CO₃•⁻) and semiquinone (SQ•⁻) [90,92,93]. These molecules play various roles in biological and chemical processes, often acting as signaling molecules or contributing to oxidative stress.

The superoxide radical is a unique molecule that acts as a reducing agent but can also give rise to powerful oxidants [90]. When it interacts with nitric oxide (NO), it sets off a chain reaction that produces other reactive molecules such as RNS, RSS, and RCS [27,28,92]. These reactive species are often seen as harmful during oxidative stress, where they can negatively affect plant growth and development. However, they are essential players in modulating redox signaling pathways, inside and outside of cells [90,91]. This means that they help maintain balance and communication within biological systems—harmful in excess but vital for normal cellular functions.

3.2. Production Sites of ROS

Reactive oxygen species are generated in several cellular organelles, including peroxisomes, chloroplasts, plasma membrane, cell wall, and mitochondria (

Table 3. Reactive oxygen species production, function, signaling, and scavenging mechanisms in plants.

Serial No.	ROS Type	ROS Production Sites	Primary Function	Cellular Reactions and Damage	Effects on Plants	Key Signaling Roles	Scavenging Mechanisms
1	Superoxide (O2•−)	Chloroplasts, mitochondria, cell membrane	Electron transport chain byproduct, signaling molecule	Damages Fe-S centers, membrane lipid peroxidation	Membrane damage, protein oxidation	Pathogen defense, cell death signaling, growth regulation	Superoxide dismutase
2	Hydrogen Peroxide (H2O2)	Peroxisomes, chloroplasts, cytosol	Cell signaling, pathogen defense	Oxidizes protein thiols, inactivates enzymes, DNA damage	Cell wall modification, root growth, stress signaling	Stress-response signaling, root growth regulation, stomatal closure	Catalase, ascorbate peroxidase
3	Hydroxyl Radical (•OH)	Cell wall, all cellular compartments	Highly reactive oxidant	Severe DNA/RNA damage, protein oxidation, cell death	DNA damage, lipid peroxidation	Programmed cell death, stress response, defense activation	Non-enzymatic antioxidants
4	Singlet Oxygen (1O2)	Photosystem II, chloroplasts	Light harvesting byproduct	Chlorophyll bleaching, lipid peroxidation, PSII damage	Chloroplast damage, PSII inhibition	Photosynthetic signaling, chloroplast-nucleus communication, stress acclimation	Carotenoids, tocopherols

). The integrated contribution of ROS from these organelles determines the total cellular ROS levels [90]. Notably, chloroplasts are the principal ROS production sites, generating 30–100-fold higher levels than mitochondria [94,95,96]. This enhanced ROS generation in chloroplasts occurs through the interaction between chlorophyll molecules and light, particularly involving the triplet chlorophyll and electron transport chains of both photosystems I and II. During photosynthesis in PSII, chlorophyll molecules within the light-harvesting complex (PSII-LHC) become activated to an energized singlet state (1Chl*), characterized by a brief lifetime of approximately ~10⁻⁸ s [97]. While a significant portion of this energy drives photosynthetic electron transport through P680 via photochemical quenching (pQ), excess energy beyond the pQ capacity dissipates through alternative pathways such as heat emission, fluorescence, or conversion to triplet chlorophyll (3Chl*) through intersystem crossing. This 3Chl* state, though lower in energy, exhibits a longer half-life of about ~10⁻³ s [97]. Under normal conditions, carotenoid pigments (specifically lutein and zeaxanthin) within the LHC effectively quench 3Chl*, preventing energy transfer to other molecules. However, the inefficient quenching of 3Chl* can lead to interactions with molecular oxygen (³O₂) released from water-splitting reactions in the oxygen-evolving complex (OEC), resulting in singlet oxygen formation [98].

In the photosystem II (PSII) reaction center, P680 undergoes photoexcitation to its singlet state (1P680*) and forms a complex with pheophytin (Pheo), resulting in 1(P680 + Pheo−) [99,100]. Subsequently, electron transfer to quinone A (QA) occurs, generating P680+ QA−. Under stress conditions where QA is pre-reduced and unable to accept electrons, 3(P680 + Pheo−) undergoes recombination with P680 to form the triplet state 3P680* [101]. Although PSII contains two β-carotene molecules capable of quenching 3P680*, their spatial separation exceeds the critical Van der Waals distance (3.6 Å), preventing effective quenching and leading to ¹O₂ formation [99,100]. Furthermore, abiotic stress-induced stomatal closure reduces chloroplastic CO₂ concentration, causing electron transport chain (ETC) over-reduction and increasing charge recombination between 1P680* and QA− in PSII, thereby enhancing ¹O₂ production [102,103]. While ¹O₂ generation is absent in PSI, ferrous iron (Fe²⁺) mediates the conversion of O₂•⁻ and H₂O₂ to hydroxyl radicals (•OH). In non-photosynthetic tissues, such as roots, mitochondrial electron leakage from complexes I and III generates O₂•⁻, which is converted to H₂O₂ by manganese and copper–zinc superoxide dismutases [103]. In peroxisomes, glycolate oxidase is the primary ROS-generating enzyme [90,103].

In peroxisomes, xanthine oxidase (XOD) generates superoxide radicals and uric acid, which are subsequently converted to hydrogen peroxide through the actions of SOD and urate oxidase [104,105]. In the peroxisome, hydrogen peroxide is produced by fatty acid β-oxidation, superoxide dismutation, and flavin oxidase activity [105]. Additionally, peroxisomal enzymes such as polyamine oxidase, copper amine oxidase, sulfite oxidase, and sarcosine oxidase also produce hydrogen peroxide [104,106]. Notably, in peroxisomes, monodehydroascorbate reductase (MDHAR) functions to neutralize hydrogen peroxide via the ascorbate–glutathione cycle [107]. The apoplastic space harbors multiple ROS-generating enzymes, including NADPH oxidase, class III peroxidases, amine oxidases, germin-like oxalate oxidases, quinone reductase, and lipoxygenases [108]. The cell wall produces ROS by peroxidases, amine oxidases, and lipoxygenases. The plasma membrane ROS generation involves NADPH oxidase and quinone reductase activities [108,109]. The endoplasmic reticulum produces superoxide via cytochrome P450, involving a complex electron transfer process [110]. Glyoxysomes produce superoxide and hydrogen peroxide via fatty acid oxidation and by the activity of glycolate oxidase and urate oxidase [18]. In the cytosol, the xanthine oxidase and aldehyde oxidase are responsible for ROS production [90,106]. In short, peroxisomes are involved in photorespiration and fatty acid oxidation, producing ROS like H₂O₂ and O₂•⁻. Mitochondria are the powerhouses of the cell, where ROS (O₂•⁻ and H₂O₂) are produced due to electron leakage from the ETC during stress. Chloroplasts are responsible for photosynthesis, where ROS (¹O₂, O₂•⁻, H₂O₂) are produced under excess light or drought stress. The endoplasmic reticulum is involved in protein folding and lipid synthesis, producing ROS (O₂•⁻, H₂O₂) during protein misfolding stress (Figure 2).

3.3. Signaling of Reactive Oxygen Species

Plants can transmit stress signals from affected tissues to healthy tissues under stress conditions. These signals trigger the defense responses and enhance resilience via signal transduction pathways, with ROS playing a central role as messengers [18,106]. However, under prolonged stress, the excessive production of ROS can cause oxidative damage and harm the cells [17]. Under these conditions, the regulation of ROS signaling pathways is important to mitigate the damage caused by stress [18,90]. Both biotic (insects/pests) and abiotic stressors can trigger ROS production in plant cells [111,112]. This disrupts the balance of ROS and activates a chain reaction of signals. These signals involve feedback and feed-forward mechanisms that help the plants adapt to stress [90,113]. The time and location of (when and where ROS production play crucial roles in how effectively the plant’s signaling networks work, which is crucial for the plant’s survival.

The plant stress response involves complex signaling cascades initiated by ROS. ROS act as cellular messengers, spreading stress signals to neighboring cells from one cell to another; this coordinated signaling effort helps the plants adapt to stress conditions. Plants use a variety of molecules, such as phytohormones, amino acids, and polyphenols, which act as signals in response to environmental stress [13,114]. During stress, ROS causes protein oxidation, creating smaller peptides that act as a secondary messenger in the signaling process. H₂O₂ (mobile molecule) is the key signaling molecule due to its non-ionic nature, relative stability, and ability to move through cellular membranes with the help of aquaporin channels [115]. ROS-mediated signaling regulates a range of cellular responses. These include activating antioxidant systems, defense-related genes, calcium ion mobilization, kinase pathways, and protein phosphorylation, all of which assist plants in withstanding stress conditions [115]. ROS signaling also increases jasmonic acid, salicylic acid, and ethylene stress hormone concentration, which play critical roles in the plant’s defense (Figure 2). During biotic stress, ROS signaling induces the production of defense proteins and phytoalexin protective compounds and reinforces the plant cell wall [116]. Additionally, ROS signaling also regulates the activity of enzymes. During stress, glutamate dehydrogenase shifts from its role in ammonia metabolism to produce the proline compound, which contributes to stress tolerance. At lower levels, ROS supports lignin production, tracheary element formation, and programmed cell death, all of which enhance plant tolerance against abiotic stress [117,118]. In short, ROS signaling is a complex and dynamic network that facilitates plants to adapt and survive under unfavorable conditions.

Under stress conditions, ROS generated in cellular organelles facilitate retrograde signaling to the nucleus at approximately 8.4 cm per minute, functioning as crucial secondary messengers in plant stress responses [109,117]. This retrograde communication facilitates the nuclear modification of anterograde signals, allowing plants to adapt to stress conditions [119]. In an unfavorable environment, the elevated ROS levels trigger the expression of stress-response heat shock genes. During temperature stress, the heat shock proteins (HSPs) are key molecular chaperones that prevent protein aggregation and assist in protein folding [109]. In addition to heat tolerance, HSPs are regulated in response to various abiotic stresses such as light, oxygen deprivation, and cold stress [120]. In Arabidopsis thaliana, cell cultures have shown that higher hydrogen peroxide induces heat shock gene expression and causes an increased activity of ascorbate peroxidase 2 (APX2) and small HSPs such as HSP17.6 and HSP18.2 [121]. In addition, the Arabidopsis H₂O₂-mediated signaling enhances ascorbate peroxidase 1 (APX1) activity, induces light stress tolerance, and improves adaptation to combined heat and drought stresses [122,123]. During hypoxia stress, respiratory burst oxidase homologs regulate ROS production, with this ROS signaling being essential for hypoxic stress management [124].

Hydrogen peroxide is an essential signaling molecule during oxygen-deficient conditions (anoxia/hypoxia). It enhances the transcription of heat shock proteins and fermentation-related genes such as alcohol dehydrogenase [125,126]. In addition, H₂O₂ modulates ZAT10 and ZAT12 transcription factors and other proteins that contribute to stress adaptation [125,126]. Reactive oxygen species demonstrate intercellular communication capabilities through wave-like propagation to surrounding tissues [117,124]. This systemic signaling mechanism, mediated by superoxide and hydrogen peroxide, has been revealed in waterlogged rice roots and Arabidopsis systems. In salt-stress responses, NADPH oxidase genes, specifically AtrbohF and AtrbohD, generate ROS that regulate cellular Na+/K+ equilibrium, thereby conferring salt tolerance [127]. Research has shown that Arabidopsis mutants deficient in AtrbohF exhibit increased salt sensitivity due to impaired ROS accumulation in root vascular tissues [127]. Under nutrient stress conditions, particularly potassium deficiency, elevated hydrogen peroxide levels in roots promote HAK5 gene expression while simultaneously activating calcium signaling pathways [128,129]. Further research into ROS-mediated molecular mechanisms and their interactions with other signaling cascades could enhance our ability to develop stress-resistant crop varieties.

4. Abiotic Stress-Induced Oxidative Stress

Plants exhibit sedentary growth patterns in natural environments [130]. However, the environmental stressors driven by anthropogenic activities and climate change trigger oxidative stress through elevated reactive oxygen species production [109]. Multiple cellular compartments are recognized as ROS generation sites (Table 3), including chloroplasts, mitochondria, peroxisomes, apoplast, and plasma membranes, with chloroplasts as the predominant source [95]. Abiotic stress typically restricts CO₂ availability, disrupting carbon fixation mechanisms. This disruption leads to oxygen reduction reactions and elevates ROS levels that compromise chloroplast function and photosynthetic efficiency [6]. ROS accumulation patterns depend on plant species, genetic makeup, stress-resistance capabilities, and exposure duration.

4.1. Heat (High Temperature) and Chilling (Low Temperature) Condition-Induced Oxidative Stress

High-temperature stress induces oxidative damage in plants through excessive ROS generation, which disrupts cellular homeostasis and leads to extensive cellular injury [79,131]. Research has demonstrated that exposure to elevated temperatures (35 °C/32 °C day/night cycles) impairs photosystem II functionality by inhibiting the reaction center and electron transport chain, resulting in decreased quantum efficiency (Fv/Fm) and compromised PSII photochemistry in rice varieties [132]. Studies in cucumber seedlings revealed that high-temperature stress (35 ± 1 °C for 7 days) triggered O₂•⁻ production, reaching nearly 80% higher than control levels [133]. In addition, malondialdehyde (MDA) levels increased by 60.6% under high-temperature stress than control cucumber seedlings [133]. Similarly, tobacco cells exposed to 50 °C exhibited a 50% elevation in O₂•⁻ levels within just 5 min of treatment, indicating the rapid onset of oxidative stress [134]. In field experiments with sorghum, high-temperature conditions (36/26 °C and 39/29 °C) caused dramatic increases in O₂^•− accumulation, with 3.5-fold and 2.3-fold higher levels detected in pollen and pistil tissues during the post-anthesis period, respectively [135]. Similarly, the extended exposure of Gossypium hirsutum to 45/30 ± 2 °C for 120 days led to a 0.78% rise in MDA content, negatively impacting fiber characteristics and boll development [136]. In Oryza sativa, heat stress at 38 °C for 5 days doubled H₂O₂ accumulation [137]. However, some studies present contrasting findings—for instance, while high-temperature treatment (38 °C for 5 days) elevated H₂O₂ levels by 27% in rice seeds, researchers observed no significant changes in O₂^•− content or lipid peroxidation markers compared to controls [137]. These studies showed that high temperature or heat stress induces ROS production, “creates redox imbalance”, and causes significant oxidative damage in various crop species.

Low-temperature stress induces excessive reactive oxygen species production in plants through several mechanisms, including compromised membrane fluidity, impaired photosynthetic machinery function, and disrupted ROS homeostasis, ultimately resulting in cellular membrane damage and electrolyte leakage [25,138]. Research has demonstrated significant oxidative stress markers in cold-stressed plants. For instance, the exposure of 14-day-old rice (O. sativa cv. DM You 6188) seedlings to cold stress (12 °C for 6 days) resulted in substantial increases in MDA levels (180%) and electrolyte leakage (49%) [139]. These findings collectively underscore the complex interplay between temperature stress and oxidative damage in plants. In another study investigating cold sensitivity, Solanum lycopersicum cv. Jinpeng No. 1 subjected to low-temperature conditions (15 °C/8 °C day/night cycle for 24–48 h) exhibited significantly higher levels of MDA and hydrogen peroxide by 62% and 34%, respectively, compared to control plants [140]. Similarly, the tomato (Lycopersicon esculentum) showed a significant increase of 32% in H₂O₂ levels under cold stress (of 4 °C for 24 h). Under cold stress, L. esculentum cv. C.H. Falat showed a 100% increase in H₂O₂ content and a 20% increase in electrolyte leakage (at 3 °C for 6 h over 6 days) [141,142]. Under progressive cold stress (4 °C for 24 h, followed by 0 °C for 12 h, and −6 °C for 12 h), comparative analysis between wild-type and transgenic Ammopiptanthus mongolicus revealed that wild-type plants showed an increased accumulation of H₂O₂ (confirmed by 3,3′-diaminobenzidine staining), indicating enhanced oxidative stress in control than transgenic lines [143]. These insights into plant responses to cold stress provide valuable directions for future research in improving stress management strategies and promoting sustainable agricultural practices under cold stress.

4.2. Drought (Water Scarcity) and Flooding (Waterlogging)-Induced Oxidative Stress

Water scarcity has a significant impact on plant cellular processes. The water deficit condition mainly disrupts photosynthesis and triggers oxidative stress [144]. Under drought stress, plants close their stomata to safe water, which limits CO₂ uptake and reduces photosynthetic efficiency. This disruption affects the light-harvesting complexes and photochemical processes in chloroplasts, leading to an overproduction of ROS [145]. In plants, several changes at the molecular level occur in response to water stress. These changes include protein denaturation, membrane damage, impaired TCA cycle enzymes, and reduced carboxylation efficiency. These alterations, coupled with reduced NADP+ regeneration, cause the electron transport chain’s over-reduction. This leads to increased electron leakage and causes excessive ROS accumulation, resulting in oxidative damage [146,147].

Different studies across various plant species have consistently shown increased ROS accumulation and oxidative damage under water deficit conditions. After 14 days of prolonged drought stress, all the wild-type Arabidopsis thaliana plants showed a more than 2-fold increase in H₂O₂, superoxide radical, MDA contents, and electrolytic leakage [57]. Correspondingly, Phragmites karka exhibited a significant increase of 22% in MDA content under 40% water-holding capacity for 35 days [148]. Coffea arabica L. also showed elevated MDA levels when exposed to prolonged drought stress for 20 days [149]. Similarly, when rice plants were subjected to 8 days of water scarcity, they showed an increase in superoxide (1.8-fold), hydrogen peroxide (2.1-fold), and MDA (1.66-fold) contents compared to control plants [150]. Finger millet under severe water deficit (75% reduction) showed significantly higher electrolyte leakage and H₂O₂ contents than the control [151]. Likewise, tomato plants exposed to 6 days of drought stress showed increased MDA content and a 39% increase in electrolyte leakage [152]. Understanding the oxidative stress regulation in plants under drought stress would be helpful to work on strategies to mitigate oxidative stress and improve drought resilience in crops.

Drought stress significantly influences oxidative stress markers in different plant species [77,144,153]. Polyethylene glycol (PEG)-induced osmotic stress in Brassica napus L. and Vigna radiata L. consistently showed enhanced ROS accumulation and membrane damage [154,155]. In soybeans, 15% PEG-induced osmotic stress significantly increased MDA, H₂O₂, and lipoxygenase (LOX) activity compared to the control soybean plants [156]. Also, tomato plants exposed to water deficit conditions (60% field capacity for 20 days) exhibited significant increases in superoxide (O₂^•−) by 75%, H₂O₂ by 37%, and MDA levels by 83% [157]. Severe drought stress in Medicago truncatula increased MDA and H₂O₂ levels; however, these parameters normalized upon rewatering [158]. The plant’s inherent genetic makeup is crucial for drought tolerance, as shown in Phaseolus vulgaris (common bean) genotypes [159]. A comparative analysis of drought-tolerant (Bn-150) and drought-sensitive (Bn-16) common bean varieties under 50% field capacity for 14 days showed that the sensitive genotype had doubled MDA content and increased ROS levels, including O₂^•−, H₂O₂, and •OH [159].

Waterlogging conditions create hypoxic or anoxic environments, mainly affecting plant roots. The hypoxia/anoxia disrupts mitochondrial respiration, leading to electron leakage from ETC (Complex I/III) and enhanced ROS (O₂•⁻ and H₂O₂) production via NADPH oxidase (RBOH) activation. The impaired antioxidant system (SOD, CAT, APX) further exacerbates ROS accumulation, triggering oxidative damage [160]. Recent research has shown significant differences in how various plant species respond to oxidative stress under waterlogging conditions. For example, a comparative analysis of waterlogging-tolerant (JN01) and -sensitive (JN31) genotypes of Sorghum bicolor revealed that the sensitive genotype has increased MDA levels compared to the tolerance genotype when exposed to waterlogging for 6, 9, and 12 days [161]. In the waterlogging-sensitive Sesamum indicum L. cv. BARI Til-4, MDA and hydrogen peroxide levels increased significantly under 2 to 8 days of flooding stress [162]. This pattern of enhanced oxidative stress markers has been consistently observed in Solanum lycopersicum [163]. Similarly, barley (Hordeum vulgare) waterlogging-susceptible TF57 showed pronounced increases in MDA content and the superoxide generation rate compared to the minimal changes in waterlogging-tolerant TF58 under 21-day waterlogging stress [164]. Notably, the Antarctic species Deschampsia antarctica exhibited elevated MDA and H₂O₂ levels under 7 days of waterlogging stress [165]. These indicate the universal nature of oxidative stress responses to waterlogging across diverse plant species.

4.3. Salt Stress-Induced Oxidative Stress

Salt stress induces several detrimental effects in plants, including ionic imbalance, osmotic pressure disruption, nutrient deficiencies, and genetic damage. These harmful effects collectively lead to enhanced ROS generation and cause severe oxidative damage in plants [166]. Under 100 mM and 200 mM, NaCl stress resulted in 2.5-fold and 3-fold increases in H₂O₂ production, respectively, accompanied by increases in lipid peroxidation [167]. The impact of salt stress varies across plant tissues and organs, with the roots generally showing the highest sensitivity, followed by older and younger leaves in a decreasing order of susceptibility. Recent studies in rice showed that salt stress doubled ROS accumulation, lipid peroxidation, and membrane damage in root tissues compared to control plants [168]. Similarly, when tomato plants were exposed to 100 mM NaCl stress, they showed significant increases in superoxide (157%), H₂O₂ (176%), lipid peroxidation (94%), and electrolyte leakage (158%) [169]. Similar responses were observed in Capsicum annuum (sweet pepper), where moderate salinity stress (0.4%) led to a 100% increase in lipid peroxidation and electrolyte leakage [170]. In addition, mung bean (Vigna radiata (L.)) plants showed a two-fold increase in H₂O₂, lipid peroxidation, membrane damage, and superoxide, under 100 mM NaCl stress [171].

Salt stress significantly increases the key oxidative stress markers, restricting plant growth and development [172]. Maize (Zea mays) plants under 120 mM NaCl stress showed increased H₂O₂ and MDA by 50% and 25%, respectively, compared to control plants [173]. The genetic background is crucial in salt stress-tolerance mechanisms, as shown by differential responses among plant species. Sunflower genotypes such as FH-572 (a salt-sensitive genotype) revealed a 78% increase in H₂O₂ levels, while FH-621 (salt tolerant) showed enhanced salt tolerance with a 20% reduction in H₂O₂, under 120 mM NaCl treatment [174]. Similarly, the TS-5 sesame varieties exhibited a higher tolerance to salt stress than TH-6 under 70 mM NaCl stress [175]. Moreover, Medicago truncatula also showed genotype-specific responses between salt tolerance and oxidative stress markers like MDA and H₂O₂, [176]. Faba bean (Vicia faba) genotypes exposed to NaCl stress showed that the hydrogen peroxide, superoxide, MDA, electrolyte leakage, and lipid peroxidation were significantly increased in all the salt-sensitive genotypes with severe oxidative damage [177]. Notably, Ailanthus altissima showed high salinity (150 mM NaCl) tolerance by enhanced antioxidant enzyme activity while maintaining H₂O₂ levels [178]. This suggests complex stress-response mechanisms for salt-stress adaptability. These findings highlight the universal nature of salt-induced oxidative stress across plant species while indicating significant variations in stress-tolerance mechanisms.

4.4. Heavy Metal Toxicity-Induced Oxidative Stress

Heavy metal toxicity in plants has a wide range of detrimental effects such as stunted growth, chlorosis, and root browning. The presence of heavy metals in soils reduces nutrient and water uptake, disrupts the electron transport chains in chloroplasts and mitochondria, and impairs peroxisomal metabolism, leading to enhanced oxidative damage [179]. The absorption of toxic metals triggers ROS levels in plants and causes significant oxidative damage in plant species [180,181]. Under nickel sulfate (0.25–0.5 mM, 72 h) stress, Oryza sativa seedlings showed enhanced lipid peroxidation and hydrogen peroxide [182]. Similarly, the pea (Pisum sativum) plants increased ROS and lipid peroxidation under mild nickel stress (100 μM NiCl₂) [183]. Cadmium stress also causes oxidative damage by accumulating malondialdehyde, H₂O₂, and superoxide in several crop species [184]. Exposure to 100 μM CdCl₂ stress resulted in a significant increase in MDA and H₂O₂ levels in Arabidopsis thaliana and cucumber seedlings [185,186]. The adzuki bean (Vigna angularis) exhibited higher lipid peroxidation, electrolyte leakage, ROS, and lipoxygenase activity under cadmium stress (100 μM CdCl₂) for 20 days [187]. Similar results were observed in Mentha arvensis under 50 µM CdCl₂ stress [188].

AlCl₃ stress in V. radiata induced an 83% increase in H₂O₂, a 110% increase in O₂•⁻, and a 72% increase in LOX activity, resulting in a 97% higher lipid peroxidation compared to control samples [189]. Under lead stress (0.5–1.0 mM Pb(NO₃)₂), wheat plants showed a severe increase in oxidative stress [190]. Moreover, pigeon pea plants exposed to arsenic toxicity (10 μM Na₃AsO₄) exhibited a 4-fold increase in lipoxygenase activity and elevated oxidative stress levels [191]. Similarly, quinoa varieties showed increased H₂O₂ and thio-barbituric acid reactive molecules under high (150–300 μM) arsenic (Na₃AsO₄) stress [192]. Copper toxicity in basil revealed a positive correlation between the copper concentration and MDA and H₂O₂ levels; as the copper levels increase, the MDA and H₂O₂ levels increase, with 1000 ppm showing the highest oxidative stress and vice versa [193]. Heavy metals (e.g., Al, Cd, As) disrupt redox homeostasis by inactivating antioxidant enzymes (SOD and CAT) and inducing Fenton/Haber–Weiss reactions, leading to excessive H₂O₂ and •OH generation. Metal-induced ROS also arise from NADPH oxidase (RBOH) activation and impaired mitochondrial electron transport [194]. To conclude, the cadmium, nickel, aluminum, and arsenic stress consistently resulted in elevated levels of hydrogen peroxide, superoxide, and malondialdehyde across different plant species. Additionally, studies revealed that even at relatively low concentrations, metals such as cadmium and arsenic can significantly trigger oxidative stress responses. This suggests a common underlying mechanism of metal-induced oxidative stress in plants, despite variations in plant species and metal types.

5. Antioxidant Enzymatic and Non-Enzymatic Scavenging of ROS and Free Radicals

Antioxidant enzymes play crucial roles in cellular defense mechanisms against ROS. SOD’s primary function involves the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen, thereby preventing hydroxyl radical formation [36]. Catalase, a tetrameric enzyme containing heme groups, detoxifies the H₂O₂. CAT exhibits remarkable efficiency in H₂O₂ degradation, converting approximately 26 million H₂O₂ molecules to water per minute [195]. Additionally, peroxidase facilitates the oxidation of phenols (PhOH) to generate phenoxyl radicals (PhO•), commonly known as quinone acceptors (QA). During this process, H₂O₂ acts as an electron acceptor and is reduced to H₂O [196]. In the absence of ascorbic acid (AsA), phenoxyl radicals undergo cross-reactions to form essential compounds such as suberin, lignin, and quinones. However, in the presence of AsA, these radicals interact with it to form monodehydroascorbate (MDHA), which further converts to dehydroascorbate (DHA) [196]. The PPO catalytic activity involves the oxidation of phenolic compounds to quinones using molecular oxygen, which significantly detoxifies ROS and produces water as a byproduct [45]. In chloroplasts, catalase is absent, and APX is the key enzyme that scavenges H₂O₂ molecules [38,195]. Moreover, the GPX enzyme uses glutathione and thioredoxins to detoxify hydrogen peroxide and several other organic hydroperoxides, including lipid peroxides. The GST enzyme performs multiple roles, such as peroxide degradation, phytohormone synthesis, stress-response signaling, and increases glutathione peroxidase activity [13]. In addition, TRX is characterized by a conserved redox-active center (WCG/PPC) that catalyzes the reduction of disulfide bonds to dithiol groups by H₂O₂. TRX showed superior efficiency compared to GSH or dithiothreitol [35]. In chloroplasts, TRX x and y isoforms maintain a cellular redox equilibrium via 2-Cysteine (Cys) peroxiredoxin reduction. In mitochondria, the TRXo1, peroxiredoxin, and sulfiredoxin collectively provide an antioxidant defense [35]. Furthermore, PRX enzymes effectively neutralize H₂O₂ and ROOH, and they require thiol cofactors (such as GSH) and have a significant ability to reduce organic and inorganic peroxides [34,35].

To conclude, plants’ enzymatic antioxidant defense system effectively quenches the ROS during oxidative stress. SOD converts superoxide to hydrogen peroxide and oxygen, CAT converts H₂O₂ into water and oxygen, peroxidases utilize different electron donors to reduce H₂O₂, and APX plays an important role in chloroplasts and cytosol [195,197]. The ascorbate–glutathione cycle enzymes such as MDHAR, DHAR, and GR maintain the reduced forms of these antioxidant enzymes [13,70]. In addition, the glutathione peroxidases, peroxiredoxins, and thioredoxins provide additional enzymatic protection by thiol-based catalysis in various cellular compartments.

Non-enzymatic antioxidants, including low-molecular-weight compounds, represent the crucial components of plant defense mechanisms against oxidative stress [75]. Ascorbate is a key electron donor in the ascorbate–glutathione cycle, maintaining stability through electron resonance between its oxidized and reduced forms [107,198]. Ascorbate regulates phytohormone pathways and regenerates α-tocopherol (from its radical form), which neutralizes free radicals (hydroxyl and superoxide). Glutathione efficiently scavenges ROS and functions as an essential regulator of cellular redox homeostasis via the ascorbate–glutathione cycle [42,199]. The antioxidant tocopherol maintains photosynthetic efficiency by protecting chloroplasts and targeting singlet oxygen and hydroxyl radicals [200]. Carotenoids provide dual protection by scavenging harmful free radicals and stabilizing light-harvesting complexes and thylakoid membranes [73,200]. Flavonoids, particularly those containing dihydroxy B-ring substitutions, demonstrate significant free radical scavenging capacity and inhibit membrane lipid peroxidation [75]. Environmental stress enhances flavonoid biosynthesis gene expression, strengthening antioxidant defenses. Phenolic compounds, including hydroxy-benzoic and -cinnamic acids, exhibit antioxidant properties through metal chelation and neutralize various reactive species [75]. Additionally, alkaloids and non-protein amino acids like GABA, ornithine, and citrulline contribute to the non-enzymatic antioxidant network through distinct protective mechanisms [69,201,202].

Non-enzymatic antioxidants such as ascorbic acid scavenge multiple ROS (O₂•⁻, H₂O₂, •OH, ¹O₂) across various cellular compartments including chloroplasts, peroxisomes, and mitochondria [198]. Glutathione functions primarily against H₂O₂ and hydroxyl radicals, while membrane-bound tocopherols and carotenoids protect against singlet oxygen and lipid peroxyl radicals in chloroplast membranes [44,198]. Secondary metabolites like flavonoids and non-protein amino acids provide additional protection by scavenging the O₂•⁻, H₂O₂, and ¹O₂ in vacuoles, chloroplast, cytoplasm, mitochondria, and cell wall. Polyphenolic compounds detoxify several reactive oxygen species such as O₂•⁻, •OH, ROO•, and ONOO– in the cell wall [75]. Some phenolic compounds have potent antioxidant activity to quench ROS and maintain cellular homeostasis under oxidative stress caused by abiotic factors (

Table 4. Specific functional roles of key polyphenols in quenching ROS under stress conditions.

Serial No:	Compound	Primary Functions During Stress	Specific Roles	Response Mechanisms	Antioxidant Activity	Modulation of Stress-Responsive Pathways	Improvement of Physiological Parameters	References
1-	Quercetin	-ROS scavenging -Membrane stabilization -Osmolyte accumulation -Metal ion chelation	-Enhances drought tolerance -Improves salt resistance -Mediates temperature stress response -Protects UV radiation	-Regulates stress-responsive genes -Enhanced enzyme activities -Aids cellular homeostasis -Membrane integrity maintenance	-Direct ROS scavenging -SOD, CAT, and POD enzyme activation -Reduced lipid peroxidation -Protection of cellular components	-ABA signaling pathway regulation -MAPK cascade activation -Calcium signaling enhancement -Transcription factor regulation	-Enhanced photosynthetic efficiency -Better water relations -Improved nutrient uptake -Increased chlorophyll content	[204,205,206,207]
2-	Kaempferol	-ROS scavenging -Membrane stability enhancement -Osmolyte regulation -Signal molecule activation -Stress-tolerance improvement	-Direct scavenging of free radicals -Prevention of lipid peroxidation -Protection of cellular membranes -Enhancement of antioxidant enzyme activities	-Upregulation of stress-responsive genes -Activation of defense pathways -Modulation of hormone signaling -Enhancement of stress-tolerance genes	-Direct free radical neutralization -Support of enzymatic antioxidants -Protection of cellular components -Reduction in oxidative damage	-MAPK signaling cascade activation -ABA-dependent pathway regulation -Stress-responsive transcription factors -Heat shock protein expression	-Enhanced photosynthetic efficiency -Enhanced growth -Better water relations -Improved nutrient uptake	[11,77,153,205,208]
3-	Luteolin	-Acts as ROS scavenger -Maintains cell membrane integrity -Enhances stress tolerance -Regulates osmolytes	-Protects photosynthetic machinery -Stabilizes cellular proteins -Reduces lipid peroxidation -Enhances membrane stability	-Activates stress-responsive genes -Induces enzymatic antioxidants -Modulates hormone signaling -Enhances proline accumulation	-Directly scavenges free radicals -Increases SOD, CAT, and POD activities -Reduces H2O2 accumulation -Prevents oxidative damage	-Activates MAPK signaling -Regulates ABA-dependent pathways -Enhances calcium signaling -Influences transcription factors	-Maintains water relations -Enhances photosynthetic efficiency -Improves nutrient uptake -Stabilizes chlorophyll content	[209,210]
4-	Apigenin	-ROS scavenging -Membrane stability enhancement -Osmolyte regulation -Signal molecule activation	-Increases antioxidant defense -Reduces reactivity of free radicals -Increases antioxidant enzyme activities -Protection of cellular membranes	-Activation of stress-responsive genes -Upregulation of heat shock proteins -Enhancement of proline accumulation -Modulation of ion channels	-Quench free radicals -Photosystem II protection -Membrane thermostability -Minimizes oxidative damage -Osmolyte accumulation	-MAPK cascade activation -ABA signaling pathway modulation -Calcium signaling enhancement -Hormone crosstalk regulation	-Improved water retention -Enhanced photosynthetic efficiency -Better nutrient uptake -Increased chlorophyll content	[210,211]
5-	Naringenin	-Acts as a potent ROS scavenger -Functions as stress-signaling molecules -Enhances membrane stability -Regulates osmolyte production -Maintains cellular homeostasis	-Drought-tolerance enhancement -Temperature stress mitigation -Salinity stress protection -Heavy metal stress resistance -UV radiation protection	-Regulates stress-responsive genes -Activation of antioxidant enzymes -Membrane lipid preservation -Ion homeostasis regulation -Photosynthetic efficiency maintenance	-Direct ROS neutralization -Antioxidant enzyme activation -Lipid peroxidation reduction -Free radical chain-breaking -Metal ion chelation	-ABA signaling modulation -MAPK cascade regulation -Heat shock protein induction -Transcription factor activation -Hormone signaling crosstalk	-Enhanced water retention -Better nutrient uptake -Improved growth parameters -Higher photosynthetic rate -Increased stress tolerance	[212,213]
6-	Ferulic acid	-Acts as a potent antioxidant -Membrane stabilizer -Osmolyte accumulation -Cell wall strengthening -Signal molecule	-Direct neutralization of ROS -Scavenges peroxyl radicals -Prevention of lipid peroxidation -Protection of cellular membranes	-Enhancement of SOD, CAT, and POD -Upregulation of stress-responsive genes -Improvement in osmolyte synthesis -Strengthening of cell wall structure	-Electron donation capacity -Free radical neutralization -Metal ion chelation -Prevention of oxidative damage	-Activation of MAPK cascades -Regulation of transcription factors -Hormonal signaling modulation	-Enhanced photosynthetic efficiency -Better water relations -Improved nutrient uptake -Increased biomass accumulation	[12,214,215,216]
7-	Chlorogenic acid	-Acts as a powerful antioxidant -Protection from oxidative damage -Maintains cellular homeostasis -Contributes to stress-tolerance mechanisms	-Scavenges reactive oxygen species -Stabilizes cell membranes -Protects photosynthetic machinery -Enhance stress-signaling pathways	-Accumulates rapidly during stress exposure -Activates defense-related genes -Triggers enzymatic antioxidants -Modulates hormone signaling	-Directly neutralizes free radicals -Reduces lipid peroxidation -Prevents oxidative damage to proteins -Maintains redox balance	-Activates MAPK signaling cascades -Induces stress-responsive transcription factors -Enhances expression of defense genes -Regulates stress hormone synthesis	-Maintains membrane integrity -Enhance water retention -Improves photosynthetic efficiency -Promotes osmolyte accumulation	[215,217]
8-	Caffeic acid	-Acts as a natural antioxidant -Precursor for lignin biosynthesis -Strengthens cell wall and its stability -Functions as a stress-signaling molecule	-UV radiation protection by absorption -Membrane integrity maintenance -Osmolyte accumulation support -Free radical scavenging -Cell wall reinforcement	-Activates phenylpropanoid pathway -Synthesized protective compounds -Cross-linking of cell wall components -Activation of stress-responsive genes	-Direct ROS scavenging -Reduction in oxidative damage -Protection of cellular components -Regulates antioxidant enzymes -Prevention of lipid peroxidation	-Regulate phenylpropanoid pathway -Activation of defense genes -Interacts with hormone signaling -Stress-responsive protein regulation -Enhanced secondary metabolites	-Better water retention efficiency -Increased membrane stability -Enhanced photosynthetic efficiency -Improved nutrient uptake -Better growth maintenance under stress	[218]

). Moreover, the O₂•⁻, •OH, H₂O₂, and ¹O₂ in the vacuoles are neutralized by alkaloid compounds [203]. This integrated network of bioactive non-enzymatic antioxidant systems protects plants against oxidative damage by direct ROS scavenging and the regeneration of antioxidant molecules.

6. Antioxidative Defense Mechanisms Under Abiotic Stress

The enzymatic and non-enzymatic antioxidant defense mechanisms work synergistically to scavenge ROS via progressive reduction reactions, ultimately converting harmful reactive oxygen species to water [33], maintaining cellular plant homeostasis, and providing critical protection under different abiotic stresses [172,203]. Under field conditions, plants frequently experience different abiotic stresses that trigger specific antioxidant responses [6]. For example, heat-tolerant Brassica juncea genotypes show enhanced resistance by regulating the peroxidase enzyme activity and increased glutathione levels [219]. Under heat stress, lentils elevated SOD, and other antioxidant activities show an inverse correlation with malondialdehyde and hydrogen peroxide levels, confirming their protective function [220]. Similarly, heat-tolerant wheat varieties (HD 2815 and HDR 77) demonstrate superior stress adaptation through the maintained activities of SOD, CAT, and APX, resulting in the better preservation of chlorophyll content and membrane integrity compared to susceptible genotypes [221]. The protective role of antioxidants extends to metal stress tolerance, as evidenced in rice plants where the coordinated activation of enzymatic (SOD, GPX, APX, GR) and non-enzymatic (ascorbate and GSH) antioxidants, along with proline accumulation, provides a defense against copper-induced oxidative stress [222]. These findings collectively determine that synchronized antioxidant responses are essential for plant survival under adverse environmental conditions.

Research has shown that when Portulaca oleracea was simultaneously exposed to heat and drought conditions, it showed elevated activities of SOD and POX enzymes [223]. The cytosolic APX1 enzyme detoxifies the H2O2 and facilitates plant adaptation against drought and high-temperature stress. Similarly, the Arabidopsis mutants lacking APX1 showed increased sensitivity to these combined stresses [224]. Carrizo citrange citrus varieties showed enhanced ROS scavenging capacity and stress tolerance against combined heat and drought stresses [225]. This stress tolerance is from efficient coordination between antioxidant enzymes SOD, CAT, APX, and GR while maintaining glutathione homeostasis. In contrast, Cleopatra mandarin showed high susceptibility under these identical stresses, caused unbalanced antioxidant responses with elevated SOD activity but reduced CAT levels, impaired GR function, and insufficient APX activity, resulting in severe oxidative damage [225].

Under drought stress, Triticum aestivum showed increased APX activity and maintained AsA and glutathione redox states, strengthening photosynthetic and mitochondrial functions [226]. Under heat stress, the Triticum aestivum-tolerant genotype C306 showed increased activities of SOD, APX, CAT, POX, and GR enzymes [227]. The Zea mays seedlings showed increased SOD and peroxidase activities when treated with nitric acid before cold exposure, resulting in reduced ROS generation and membrane lipid peroxidation [228]. When exposed to copper stress, Oryza sativa exhibited elevated activities of SOD, GP, APX, and GR, along with increased AsA, GSH, and proline levels, while maintaining stable CAT activity [222]. This resulted in decreased hydrogen peroxide accumulation and reduced lipid peroxidation. Salinity-stressed Oryza sativa seedlings treated with manganese showed enhanced levels of both non-enzymatic (phenolics, flavonoids, AsA) and enzymatic (monodehydroascorbate reductase, dehydroascorbate reductase, SOD, CAT) antioxidants [229].

Plants exhibit diverse antioxidant responses when subjected to combined abiotic stresses. Triticum aestivum seedlings under concurrent low temperature and isoproturon stress showed enhanced antioxidant activity following AsA application [230]. Similarly, Oryza sativa exposed to combined salinity and 2,4-dichlorophenoxyacetic acid stress exhibited modulated enzymatic and non-enzymatic antioxidant responses, effectively reducing oxidative damage [231]. In Cucumis sativus exposed to chilling stress, enhanced activities of SOD, APX, GR, and GPx were observed, while CAT activity decreased in leaf tissues [232]. Similarly, UV-B radiation exposure in Helianthus annuus cotyledons increased CAT, glutathione dehydrogenase, and GP activities, with elevated GSH/GSSG ratios [233]. In Spinacia oleracea seedlings, the ectopic expression of cytosolic heat shock protein 70 (SoHSC70) enhanced antioxidant enzyme activities, effectively reducing oxidative damage and ROS accumulation [234]. Artemisia annua increased the activities of SOD, POX, and CAT enzymatic antioxidants in response to boron stress [235]. The transgenic Arabidopsis thaliana plants showed an increase in non-enzymatic antioxidants such as flavonoids and anthocyanins with lower ROS, under drought and high light stress [57,58]. We summarize the intricacy of plant enzymatic and non-enzymatic antioxidants working together to maintain cellular homeostasis under environmental stress, as well as plant survival in hostile conditions.

7. Conclusions

This review summarizes the enzymatic and non-enzymatic antioxidants that work in concert to neutralize the ROS and maintain the cellular redox homeostasis under oxidative stress. The key enzymatic antioxidants such as SOD, CAT, APX, and GR form the first line of defense by directly neutralizing the ROS. Non-enzymatic antioxidants such as ascorbate, glutathione, and bioactive polyphenols, including flavonoid compounds, provide additional protection through their radical scavenging abilities. The production and accumulation of ROS in different cellular organelles serve dual functions—as important signaling molecules at lower concentrations and as damaging oxidative agents (at higher concentrations) under severe stress conditions. Understanding these context-dependent functions is crucial for developing stress-tolerant crops. Our review revealed that the oxidative stress response shares common elements under various abiotic stresses, and each stress type triggers specific antioxidant responses depending on the nature and severity of the stress.

ROS can boost plant stress tolerance when managed strategically. Seed priming with mild ROS (like H₂O₂ or NO) activates defense genes, while antioxidants (ascorbic acid and glutathione) prevent oxidative damage. Tailoring these methods to crop needs, such as appropriate timing and crop-specific dosage, can improve the resilient and sustainable path to climate-smart agriculture. Recent advances in understanding ROS signaling networks and antioxidant systems will be invaluable for engineering stress-resistant crops with enhanced adaptability to changing environmental conditions, ultimately contributing to global food security.