Introduction

Plants constantly adjust their metabolism to match their environment. Three inherently oxidative processes are the core of plant metabolism: photosynthesis, photorespiration, and oxidative respiration, all three are connected by the same cellular pools of reductants and antioxidant enzymes (reviewed in Foyer and Noctor 2000; Noctor et al. 2007). The formation of reactive oxygen species (ROS) is an unavoidable consequence of oxidative metabolism, which takes place to a greater or lesser extent in every cell type. Typically, these effects are balanced by cellular reductant pools, which are themselves regenerated by the same processes. Small changes in the environment can quickly cause a change in this delicate redox balance, resulting in a ROS burst. For example, a breeze moving the overhead canopy will allow full sunlight to hit an understory leaf and quickly lead to a sharp spike in the absorption of light energy by pigments of the photosynthetic antennae, which then causes a local ROS burst in the chloroplast (Li et al. 2009). In this context, ROS bursts are valuable as cellular signals of environmental changes. They can trigger plant acclimation and are an established part of plant sensing and response mechanisms (Choudhury et al. 2017; Farooq et al. 2019). When long-term adverse conditions occur, such as drought, salt stress, temperature shifts, predation, or nutrient limitation, damaging levels of ROS can accumulate either directly, or because of the metabolic shifts required for acclimation (Ensminger et al. 2006; Dumanović et al. 2021).

All photosynthetic organisms are powered by the oxidation of water using light energy and the subsequent photosynthetic electron transport chain. At the core of photosystem II (PSII) are two closely paired chlorophyll molecules that work as a single unit (Raszewski et al. 2008). Together in the physical arrangement conferred by their photosystems, they represent the most oxidizing molecules in biology (Rappaport et al. 2002). The oxidation of chlorophyll is also light driven and thus exquisitely sensitive to light fluctuations that inevitably occur each day (Ruban 2009). To maintain redox homeostasis, the variation in total redox capacity of PSII must be appropriately buffered to avoid damage to cellular macromolecules such as proteins, lipids, and nucleic acids. To counterbalance the strong oxidation potential in PSII, the entire plant redox metabolism has evolved to include multiple, layered tiers of reductant and antioxidant enzymatic systems.

Endogenous plant antioxidant systems help modulate ROS signaling and mitigate ROS damage. These systems typically include both chemical reductants and enzymes that can detoxify ROS or regenerate the reductants (Gill and Tuteja 2010). Reductants are normally maintained in a highly reduced state, except in the vacuole and in extracellular spaces, where they cannot be efficiently regenerated (Schwarzlander et al. 2008; Karpinska et al. 2018). Antioxidant systems contribute to environmental sensing, respond to stresses, and are linked to plant defense systems (Gechev et al. 2006). Because antioxidants provide natural stress resistance and pathogen defense, there is interest in harnessing antioxidant systems to increase crop yield and resilience (Arnao and Hernandez-Ruiz 2015; Zhu et al. 2017; Broad et al. 2020). Species with altered antioxidant levels have been bred for better crop health, increased medicinal value, and natural pest resistance.

Notably, the exogenous application of antioxidants constitutes a direct route to improve plant health and resilience. A similar use of exogenous antioxidants in mammalian and microbial systems is well established (Takagi et al. 1997; Smeriglio et al. 2016). In plants, investigations of antioxidant applications in numerous species have indicated that they can improve plant health and change native antioxidant systems, which suggests a strong potential for their future agricultural applications. Antioxidant application would offer a faster means of deployment in the field than plant genetic engineering because they require relatively less time to optimize, should be non-toxic or even beneficial to humans and mammals at the levels applied, and may avoid the controversy associated with genetically engineered crops. Importantly, they may also avoid the typical growth–defense trade-off. Indeed, plants engineered to exhibit increased defense responses tend to suffer from reduced growth and vice versa (Huot et al. 2014; van Butselaar and Van den Ackerveken 2020). Both increased growth and stress tolerance in a diversity of plant species have been observed after the application of a wide range of different antioxidant compounds. In this review, we describe multiple antioxidants and their effects on plant stress resilience, photosynthesis, endogenous antioxidant systems, and phytohormones. Our goal is to explore the metabolic and physiological effects of antioxidant applications and identify common patterns between different chemical compounds. We conclude by highlighting important target areas of future research for agricultural use of antioxidants and for determining the mechanistic details of the effects of their application.

Individual antioxidants

Before delving into a detailed analysis of the reported effects conferred by the exogenous application of antioxidants to plant health and development, we will briefly introduce the major chemicals discussed in this review and any endogenous roles they have in plants to lend perspective to later discussions. We have chosen these antioxidants because of their ability to act as chemical reductants (glutathione, ascorbate, N-acetylcysteine, melatonin, tocopherol, thiamine, flavins, and chitosan), their similar chemistry and activation of endogenous antioxidant systems (proline, GABA, allantoin, and benzothiadiazole), and the frequency of reported exogenous application.

Glutathione

Reduced glutathione (GSH) is a naturally occurring, low molecular weight thiol tripeptide formed by glutamate, cysteine, and glycine (structure in Fig. 1). The thiol (-SH) group from cysteine makes it redox-active. Glutathione is the most abundant cellular thiol in most organisms, including the model plant Arabidopsis (Arabidopsis thaliana), and it is present in high concentrations in mitochondria, the cytosol, chloroplasts, nuclei, and peroxisomes (Zechmann et al. 2008; Gill et al. 2013). Glutathione is also present at relatively high levels in the phloem, suggesting that it can be transferred from cell to cell and redistributed throughout the plant (Mendoza-Cozatl et al. 2008). As a major cellular antioxidant, GSH has multiple functions, including roles in redox regulation, defense, growth, development, the cell cycle, cell proliferation, metabolism, gene expression, and protein activity, all of which have been reviewed elsewhere (Noctor et al. 2012; Mhamdi et al. 2013; Hasanuzzaman et al. 2017).

Fig. 1
figure 1

Structures of small molecules discussed in this review. Structures appear in the same order as they occur in the “Individual Antioxidants” section. α-Tocopherol, riboflavin, the flavone backbone, and benzothiadiazoles are representative structures from a structurally related group of molecules. Irregular chitosan modifications are not illustrated. Redox potentials are given when available (Lundblad and Macdonald 2018)

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GSH is readily oxidized to GSSG, so cells maintain most glutathione as GSH by glutathione reductase enzymes (Mahmood et al. 2010). The overall glutathione pool and the relative ratio of GSH to GSSG are critical for many physiological processes, including maintaining photosynthetic efficiency (Müller-Schüssele et al. 2020) and conferring stress tolerance (Dubreuil-Maurizi et al. 2011; Dubreuil-Maurizi and Poinssot 2012; Koffler et al. 2014). Glutathione’s role in photosynthesis can be attributed in part to its ability to reduce ascorbate, a co-substrate in the production of a carotenoid required for non-photochemical quenching (NPQ, Fig. 2, Arnoux et al. 2009). Glutathione’s role in stress response is likely partly due to its important role in limiting lipid peroxidation and membrane damage, a common occurrence during exposure to multiple stresses including cold (Orvar et al. 2000), drought (Kumar et al. 2015), salinity (Zhu 2002), and heat (Niu and Xiang 2018). The increased stress tolerance observed following the exogenous application of GSH is likely due to these and additional underlying rationales for each stress (reviewed in Hasanuzzaman et al. 2017).

Fig. 2
figure 2

Effects of exogenous antioxidant applications on the light reactions of photosynthesis, non-photochemical quenching, and the Calvin-Benson-Bassham cycle. A. Relative fluxes through non-photochemical quenching (NPQ), photosystem II (PSII), photosystem I (PSI), and the Calvin-Benson-Bassham (CBB) cycle during stress conditions. Stresses such as high light and cold temperatures cause an increase in the proton gradient and lumen acidification. As a result, reaction centers become over-reduced, photosystems are damaged, the protective mechanism of NPQ is increased (bold, blue arrow), and generation of reactive oxygen species (ROS) is increased (bold, blue arrow). Electron fluxes and carbon assimilation rates are low, indicated by thin lines. B. A generalized effect of antioxidant application on NPQ, PSII, PSI, and the CBB cycle during stress conditions. Increased levels are indicated by bold font and blue arrows. Applied antioxidants can directly quench ROS, enhance electron flow through the Mehler reaction, or convert violaxanthin (Vx) to zeaxanthin (Zx) through violaxanthin de-epoxidase (VDE), which uses ascorbic acid (ASC) and modifies NPQ. A question mark indicates the variable effect on NPQ. Increased abundance of electron transport and carbon reaction rates is indicated by thicker lines. ADP, adenosine diphosphate, ATP, adenosine triphosphate; Chl, chlorophyll; Cyt b6f, cytochrome b6f; Fx, ferredoxin system; H+, proton; NADP+, nicotinamide adenine diphosphate; NADPH, reduced nicotinamide adenine diphosphate; PC, plastocyanin; Pi, inorganic phosphate; PQ, oxidized plastoquinone; PQH2, reduced plastoquinone; ZEP, zeaxanthin epoxidase; PsbS, photosystem II protein S, a molecular component of NPQ

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Ascorbate

Ascorbate is an α-keto-lactone (structure in Fig. 1) that can reduce singlet oxygen, superoxide anions, hydroxyl radicals, and hydrogen peroxide through an ene-diol group on its lactone ring (Shamberger 1984; Smirnoff 1996). Like GSH, ascorbate is a chemical reductant central to plant metabolism. Ascorbate is the most abundant antioxidant identified in the apoplast, where it detoxifies ozone, as recently reviewed (Bellini and De Tullio 2019). Ascorbate is required for the biosynthesis of key phytohormones that regulate development and stress responses, including abscisic acid (ABA), gibberellins (GAs), and ethylene (reviewed in Barth et al. 2006; Ye et al. 2012; Zhang et al. 2013). In addition to quenching ROS, ascorbate is frequently used to regenerate other antioxidants (e.g., Fig. 3, Gallie 2013; Smirnoff 2018).

Fig. 3
figure 3

Plant endogenous antioxidant systems. A. Subcellular locations of key antioxidant systems in plants are shown. Peroxisomes, mitochondria, chloroplasts, and cytoplasm each have complete glutathione (GSH) ascorbate (ASC) cycles, shown in the most detail in the cytoplasm for space. Metabolism occurring in peroxisomes, mitochondria, and chloroplasts generates superoxide (O2*−), which is typically short-lived and converted to hydrogen peroxide (H2O2), which is longer lived and can diffuse through membranes, indicated by dashed lines. The hydrogen peroxide is detoxified by one or more of the mechanisms shown at right, catalase (CAT), glutathione peroxidase (GPX), and ascorbate peroxidase (APX). Oxidized glutathione is recovered by glutathione reductase (GR), and oxidized ascorbate by dehydroascorbate reductase (DHAR, shown), or monodehydroascorbate reductase (not shown, presence indicated by an asterisk). The reactions of these enzymes are illustrated in the cytoplasm for convenience; however, they occur in multiple compartments indicated by their superscripts (Pe, peroxisome; V, vacuole; C, cytoplasm; Pl, plastids including chloroplasts; M, mitochondria). B. Apoplast antioxidant system. Under conditions of extracellular damage and biotic and abiotic stress, respiratory burst oxidase homolog (RBOH) enzymes produce ROS to signal stress and defend against pathogens. Most apoplastic ROS are converted to hydrogen peroxide through the action of superoxide dismutase (SOD). Peroxidases, both soluble and bound to the cell wall, generate ROS to allow cell elongation and to amplify stress signals. The ROS balance in the apoplast influences both plant growth and stress responses. ASC reacts with hydrogen peroxide to form water through the action of APX. ASC is converted to monodehydroascorbate (MDHA) or dehydroascorbate (DHA) and returns to the cytoplasmic ASC GSH cycle to be ultimately reduced by NADPH as illustrated in A. Extracellular antioxidants may directly interact with apoplastic hydrogen peroxide or regenerate ASC

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Amino acids

In plants, free amino acids have essential roles in mineral nutrition, signaling, and modulation of redox homeostasis (Hildebrandt et al. 2015). When applied exogenously, certain amino acids have been shown to confer greater tolerance to many stressors, such as drought, heavy metals, and salt in a variety of plant species, including Arabidopsis, wheat (Triticum aestivum), rice (Oryza sativa), and maize (Zea mays). Here, we consider a subset of amino acids with innate antioxidant properties or those that trigger antioxidant responses in plants, specifically N-acetylcysteine, proline, and γ-aminobutyric acid (Table 1).

Table 1 Effects of exogenous antioxidant application on plant growth during stress
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N-acetylcysteine

N-acetylcysteine (NAC) is a derivative of cysteine and a precursor of glutathione (structure in Fig. 1) that has antioxidant properties as well as therapeutic and clinical applications (Dhouib et al. 2016). The antioxidant and radical scavenging mechanisms of NAC are not fully understood, although a recent review discussed multiple possibilities (Aldini et al. 2018). First, NAC is a precursor of GSH, suggesting that it may raise overall antioxidant metabolism partly because it contributes directly to the glutathione pool. Second, NAC can directly reduce radicals such as nitrous dioxide (NO2) and hypohalous acids (HOX) via its thiol group. Finally, NAC can also resolve intra-molecular and inter-molecular disulfide bonds in thiolated proteins, a property that may contribute to its antioxidant effects by freeing thiols (Aldini et al. 2018).

Proline

The proteinogenic amino acid proline is oxidized to glutamate in mitochondria (structure in Fig. 1). Its ring structure makes it an excellent osmolyte, and it affects redox homeostasis in multiple organisms, although it is not itself a reductant. Proline accumulates to relatively high concentrations in plant tissues during various types of stress conditions (Szabados and Savoure 2010) and has therefore been intensively studied. Beyond its redox effects, proline has been proposed to act as a metal chelator, an intracellular osmolyte, and an amino acid metabolite that positively influences plant abiotic stress responses (Hayat et al. 2012; Liang et al. 2013; Fichman et al. 2015; Gulyas et al. 2017). Synthesis, sensing, signaling, transport and physiological functions of proline have recently been reviewed (Hosseinifard et al. 2022).

GABA

γ-Aminobutyric acid (GABA) is a prominent non-proteinogenic amino acid ubiquitously distributed across all domains of life (Bown et al. 2006). It is not a chemical reductant (structure in Fig. 1); however, GABA accumulation controls redox homeostasis in multiple organisms (Allan et al. 2008; Tang et al. 2018). In plants, GABA primarily forms from glutamate (Yang et al. 2015) and is involved in nitrogen metabolism (Bouche and Fromm 2004), signaling, plant growth and development (Fait et al. 2008; Renault et al. 2011), and stress response (Kinnersley and Turano 2000; Podlesakova et al. 2019). GABA accumulates under various stress conditions and is triggered by cytosolic pH acidification or calcium (Ca2+) signaling, which lead to the activation of the GABA biosynthesis pathway (Kinnersley and Turano 2000).

Melatonin

Melatonin (N-acetyl-5-methoxytryptamine, structure in Fig. 1) is a powerful antioxidant capable of broadly scavenging up to 10 ROS per molecule and is ubiquitous to all organisms (Posmyk and Janas 2009; Reiter et al. 2016). There are four known biosynthetic pathways for melatonin in plants, all of which begin with tryptophan and utilize serotonin as an intermediate (Back et al. 2016). After biosynthesis, melatonin acts as both an antioxidant and a phytohormone (reviewed in Fan et al. 2018); it is critical to growth and multiple developmental processes including the promotion of photosynthesis, chlorophyll preservation, delaying leaf senescence, and redox homeostasis (Tan et al. 2012; Arnao and Hernandez-Ruiz 2014). A unique melatonin receptor was recently identified in plants (Wei et al. 2018), and it is hypothesized to be responsible for increased calcium signaling, as the expression of calmodulins is induced upon melatonin treatment (Shi et al. 2015; Yang et al. 2021a). In a possible sequence of events, two mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which are primarily associated with pathogen resistance, are strongly and transiently activated in response to melatonin application on both Arabidopsis and tobacco (Lee and Back 2016). Ultimately, melatonin induces widespread changes in expression of genes encoding phytohormone signaling components, transcription factors, and ROS scavenging enzymes, which has led to the suggestion that it plays a central role as an endogenous plant antioxidant (Hardeland 2016).

Tocopherols

Tocopherols, also known collectively as vitamin E, are synthesized in plants, algae, and some cyanobacteria. In green tissues, the predominant form of vitamin E is α-tocopherol (structure in Fig. 1), which together with carotenoids forms the most abundant group of lipid-soluble antioxidants present in chloroplasts (Havaux et al. 2005). Their reaction produces a tocopheroxyl radical that is recycled to tocopherol again by reacting with ascorbate (Munne-Bosch 2005). The best known functions of tocopherols are protection of membrane lipids from peroxidation, detoxification of lipid peroxides, and quenching of singlet oxygen, which are achieved through multiple mechanisms (Suarez-Jimenez et al. 2016; Munoz and Munne-Bosch 2019). Plants lacking tocopherols exhibit greater lipid oxidation during seed desiccation and quiescence, poor seed germination, and substantially shorter roots at maturity (Mene-Saffrane et al. 2010), indicating their importance for plant growth and survival.

Thiamine

Thiamine, also known as vitamin B1, is the unphosphorylated form of thiamin pyrophosphate (structure in Fig. 1), a co-enzyme with multiple nitrogen and sulfur-containing rings (Goyer 2010; Colinas and Fitzpatrick 2015). Thiamine serves as a chemical reductant capable of transferring two protons and electrons, through one or more mechanisms (Lukienko et al. 2000; Nga and Quang 2019). As a reductant, thiamine can directly react with ROS in the form of hydroxyl radicals and superoxide (Hu et al. 1995; Jung and Kim 2003). Endogenous thiamine levels increase in response to multiple stresses such as salinity or cold, possibly due to its need as a cofactor, as thiamine pyrophosphate is a required co-enzyme for several reactions of central metabolism.

Allantoin

Allantoin is a nitrogen-rich compound released during purine breakdown in peroxisomes (structure in Fig. 1, Lamberto et al. 2010). It has been suggested to act as a long-distance nitrogen transport molecule in plants (Fahad et al. 2019). Allantoin directly reacts with H2O2 (Gus'kov et al. 2002) and appears to have ROS-protective effects in both plants and bacteria in vivo through activation of native plant antioxidant enzymes (Gus'kov et al. 2002; Irani and Todd 2016; Nourimand and Todd 2016). However, endogenous allantoin concentrations are lower than canonical plant antioxidants, as they are below 1 μmol per g of fresh weight in Arabidopsis (Brychkova et al. 2008). Furthermore, allantoin cannot scavenge free radicals, chelate ferrous iron (Fe2+), or inhibit fatty acid oxidation (Wang et al. 2012a). Thus, the extent to which allantoin acts as a direct reductant instead of an activator of plant antioxidant systems remains unclear.

Flavins and flavonoids

Flavins, characterized by a modified isoalloxazine ring (Voet and Rich 1971), are a widespread class of natural products that serve as nutrients, antioxidants, and electron carriers and are synthesized by all organisms. Common examples include flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (structure in Fig. 1). Both FMN and FAD are enzyme cofactors that contribute directly to the photosynthetic and mitochondrial electron transport chains. Studies of mutants defective in flavin biosynthesis have been especially useful in ascribing roles for flavins to physiological processes including electron transfer and central metabolism (Hedtke et al. 2012; Hiltunen et al. 2012). In addition to promoting growth and affecting plant metabolism, riboflavin induces resistance to fungal, bacterial, and viral pathogens (Aver'yanov et al. 2000; Dong and Beer 2000; Zhang et al. 2009; Taheri and Tarighi 2010; 2011).

Flavonoids are phenolic chemicals that roughly mimic the isoalloxazine ring of flavins (Panche et al. 2016), although they present more diverse chemistries. Major classes of flavonoids are flavones (structure in Fig. 1), flavonols, isoflavones, chalcones, and anthocyanins (Rice-Evans et al. 1996; Ali et al. 2016; Panche et al. 2016). Flavonoids can scavenge antioxidants (Agati et al. 2012; Ali et al. 2016; Song et al. 2020; Ummat et al. 2020), and their antioxidant applications include medicinal use in humans (Lee et al. 2011; Baselga-Escudero et al. 2014; Choy et al. 2016; Khan and Kamal 2019; Raman et al. 2019). These abundant phytochemicals are responsible for many of the colors, flavors, and fragrances of plants and can have diverse structures and chemical activities (Li et al. 2019). Mutant studies suggest that flavonoids are integral to plant development and physiology. Anthocyanins help attenuate the effects of high light stress (Zheng et al. 2020). Quinones limit the toxicity of endogenous polyphenols and cytokinin biosynthesis and are important for growth and gametogenesis in the moss Physcomitrium patens (Richter et al. 2012). Flavonoid contents and polyphenol oxidase activity were shown to correlate with increased plant vigor in red clover (Trifolium pratense L. cv. Milvus), independently of their antioxidant properties (Boeckx et al. 2015, 2017). The effect of exogenous flavonoid application to plants is not established, though their relationship to flavins, their antioxidant properties, and their application as antioxidants in other systems suggest that they might have positive effects.

Chitosan

Chitosan is a pseudo-natural cationic polymer composed of randomly distributed amide derivatives of glucose linked in β-1,4 configurations (generalized structure in Fig. 1). Chitosan is derived from the deacetylation of chitin, a component of arthropod exoskeletons and fungal cell walls (Rinaudo 2006; Prashanth and Tharanathan 2007). Small soluble chitosan polymers have antioxidant properties that can protect lipids, DNA, and proteins from oxidation (Liu 2008; Ngo et al. 2009), presumably from the available reducing sugar (C1). Chitosan is not an endogenous molecule in plants and has been applied to them in part because of its similarity to cell wall components of plant pathogens (reviewed in, Chandra et al. 2015; Chakraborty et al. 2020). The positive effects of chitosan application may stem from these defense-related responses. Importantly, studies have yet to separate the activation of defense responses from the antioxidant properties of chitosan in plants (Hidangmayum et al. 2019).

Benzothiadiazole

Benzothiadiazole (BTH), also known as acibenzolar-S-methyl, was initially discovered as a compound that activated systemic acquired resistance against pathogens (Gorlach et al. 1996) and was commercialized for this ability. BTH is sold commercially under the name Actigard® in the USA (Schurter et al. 1997) and BION® in Europe (Kunz et al. 1997; Oostendorp et al. 2001). Because it activates salicylic acid (SA) receptors in Arabidopsis SA biosynthetic mutants, BTH is commonly considered a functional SA analog (Lawton et al. 1996; Ryals et al. 1996). It is typically applied at levels consistent with those of other antioxidants presented in this review and has a conjugated ring system that allows it to act as a reductant (structure in Fig. 1, Neto et al. 2013). It is currently unclear to what extent BTH action reflects its antioxidant properties. A transcriptome analysis of purple false brome (Brachypodium distachyon) comparing SA and BTH application determined that 1,339 genes were expressed in response to BTH and not SA (Kouzai et al. 2018a). Also in B. distachyon, applications of BTH lead to unique effects distinct from those observed following SA applications (Kouzai et al. 2018a, 2018b), suggesting that BTH- and SA-mediated effects are separable. Distinguishing between the phytohormone-like and antioxidant properties of BTH would be an interesting avenue for future studies.

Effects of antioxidants: from the cellular to the environmental scale

Having introduced the antioxidants we cover, in the next section, we catalog the reported effects of antioxidant application on plant stress resilience which are broadly positive. We then probe common patterns of response to antioxidants in three commonly reported areas of plant metabolism: photosynthesis, endogenous antioxidant systems, and phytohormones.

Effects on stress tolerance

Stress, whether biotic or abiotic, causes shifts in metabolism that result in a temporary ROS accumulation. ROS, in turn, are often potent chemical messengers that trigger cellular stress responses (Choudhury et al. 2017; Farooq et al. 2019). Further, environmental stresses often occur simultaneously and can have similar effects on plant physiology (Mittler and Blumwald 2010). Examples include cellular-level dehydration associated with freezing, high salt, and drought stress or the overloading of the light-harvesting machinery associated with high light or low temperature. Plant responses to distinct stresses therefore exhibit considerable transcriptional, signaling, and metabolic overlap that together coordinate protection of cellular membranes, photosynthesis, and more broadly metabolism (Kreps et al. 2002). Primary messengers of plant stress tend to be distinct, but secondary stress messengers are often shared by multiple stress pathways. For example, calcium and ROS are common stress secondary messengers (Suzuki et al. 2012; Gilroy et al. 2016; Fichman and Mittler 2020). These secondary messengers then directly or indirectly trigger transcriptional changes through a set of conserved transcription factors, many of which are common to two or more stress conditions. Finally, plant growth is affected by stress through the modulation of plant hormones (see Effects on Phytohormones) and changes to photosynthesis (see Effects on Photosynthesis).

Because exogenously applied antioxidants will likely diminish reactive oxygen signals used to communicate stress, it is perhaps surprising that so many antioxidants nevertheless provide growth benefits during multiple stresses (Table 1). One hypothesis to explain this discrepancy is that low levels of antioxidants are not sufficient to interrupt reactive oxygen signaling and prevent effective acclimation. Some potentially supporting evidence is that high doses of antioxidants inhibit growth in Arabidopsis (Qian et al. 2014). Possible mechanisms for the growth improvement observed upon the application of antioxidants during stress include changes to phytohormones, native antioxidant pools, and photosynthesis rates discussed in detail below (Chen et al. 2012; Ejaz et al. 2012; Ding et al. 2016; Irani and Todd 2018).

The diversity of chemical structures that can provide improved growth during salt, drought, temperature variation, nutrient limitation, and presence of heavy metals (Fig. 1, Table 1) suggests that specific chemical features are less relevant than their intrinsic antioxidant properties in conferring tolerance. For example, glutathione, proline, and ascorbate improve tolerance to cadmium in barley (Hordeum vulgare), rice, and maize (Wang et al. 2011; Cao et al. 2015; Zhang et al. 2019a); iron deficiency in Arabidopsis and cucumber (Cucumis sativus) (Ramirez et al. 2013; Guo et al. 2020); salt in Arabidopsis, maize, and tobacco (Nicotiana tabacum); drought tolerance in Arabidopsis, maize, and B. napus (Chen et al. 2012; Shafiq et al. 2014; Altuntas et al. 2020); and low temperature tolerance in maize, tomato (Solanum lycopersicum), and quinoa (Chenopodium quinoa) (Pei et al. 2018; Yaqoob et al. 2019; Elkelish et al. 2020). A unifying mechanism may rely on their antioxidant properties as being responsible for the observed effect, either by supplementing endogenous ROS scavenging mechanisms or by affecting native antioxidant levels. Alternatively, individual chemical properties may converge to provide tolerance, although the lack of comparative studies between these antioxidants precludes further comment on this possibility.

Table 1 also indicates gaps in the current literature when antioxidant applications might demonstrate a growth benefit, for example, proline or GABA during heat stress, glutathione during hypoxia, most antioxidants during direct oxidative stress such as ozone exposure, and tocopherols, thiamine, and chitosan during heavy metal stress. Another gap in knowledge is the apparent distinction between biotic and abiotic stress. While native (endogenous) glutathione is well known to play an important role in resistance against fungal and bacterial plant pathogens (reviewed in Hernández et al. 2017), exogenous applications of glutathione have not been tested in the context of any biotic stressors. Conversely, the effect of ascorbate, amino acids, and melatonin applications have all been explored for their ability to improve abiotic stresses, although GABA, NAC, and melatonin applications have also been shown to improve outcomes against pathogens (biotic stress) (Muranaka et al. 2013; Fu et al. 2017; Yang et al. 2017; Zhao et al. 2019). By contrast, studies on the role of flavins and BTH have focused on biotic stress but also display some potential for abiotic stress tolerance (Jespersen and Huang 2017; Jespersen et al. 2017). Therefore, there is a clear crossover in the effects of antioxidants against both biotic and abiotic stress factors; future studies exploring the benefits of applications known to provide resistance to abiotic stress for biotic challenges and vice versa would be worthwhile.

The concentrations of applied antioxidants are critical to their ability to impact plant health, as noted in a review of melatonin’s role in stress tolerance (Zhang et al. 2015a). All antioxidants require high enough levels to show a positive effect on growth, and high concentrations of ascorbate, melatonin, allantoin, tocopherol, flavins, chitosan, and BTH were shown to cause negative growth impacts (Table 1). For example, in Arabidopsis, high concentrations of ascorbic acid (Qian et al. 2014), proline (Nanjo et al. 2003), and melatonin (Hernández et al. 2015; van Hulten et al. 2006) negatively impact growth. Response curves to antioxidant application have also been observed in pistachio (Pistacia vera) after ascorbate application (Bastam et al. 2013), in apple (Malus domestica) during drought after GABA application (Liu et al. 2021), in cherry (Prunus avium and Prunus cerasus) after melatonin application (Sarropoulou et al. 2012), in sugar beet (Beta vulgaris) after allantoin application (Liu et al. 2020), and in basil (Ocimum ciliatum and Ocimum basilicum) after application of chitosan (Kim et al. 2005).

Effective concentrations of antioxidants useful for promoting plant health vary based on developmental stage, cultivar, and environmental conditions. One example of the dependence on developmental stage, the concentrations of melatonin that improve growth of Arabidopsis when applied to germinating seeds is much higher than for plants in their reproductive stages (Hernández et al. 2015). The importance of the environment is shown by many studies that compare antioxidant treatement under a stress and control conditions (Bastam et al. 2013; Zhou et al. 2021; Elkelish et al. 2020; Rahman et al. 2020; Saeidi-Sar et al. 2013). In two typical examples, the same level of ascorbate applied to improve salt tolerance under control conditions improved growth less, not at all, or negatively in multiple genotypes of rice (Rahman et al. 2020) and beans (Saeidi-Sar et al. 2013). Based on published studies, the most frequent reports of negative growth impacts resulting from antioxidant applications occur during non-stress conditions (Table 1). We can speculate that this occurs from the common publication bias for positive, statistically significant results and that negative growth effects are simply underreported. It is impossible to know if this is the case. However, the presence of clear affect curves for most antioxidants and the dependence of concentration on developmental stage also argues that finding the effective antioxidant concentration is application-dependent and not trivial. A systematic dissection of the response to increasing amounts of antioxidants may help us better understand the the linkage between growth effects and molecular mechanism(s) underlying ROS homeostasis.

At least three antioxidants do not appear to cause any negative growth effects when applied exogenously at high concentrations, glutathione, amino acids, and thiamine (Table 1). There are possible rationales why each of these may never appear to show toxic effects. Glutathione and amino acids are catabolized with high efficiency. Glutathione breakdown has been estimated at 30 nmol per gram of fresh weight per hour (Ohkama-Ohtsu et al. 2008), a rate that would allow complete degradation of cellular glutathione within a few hours (Noctor et al. 2012). Similarly, there is an efficient interconversion system for most amino acids, including proline and GABA (Kishor et al. 2005). Thimine appears to have a strong negative feedback loop that is activated by external thiamine application. For example, when thiamine is applied to date palms, the internal thiamine levels decrease (Subki et al. 2018).

In summary, exogenously applied antioxidants typically improve growth in a variety of plants under multiple stress conditions. The effects observed under stress are typically larger than those observed under normal conditions, where application can be detrimental. We speculate that growth impacts occur because of changes to native antioxidants, phytohormones, and photosynthesis, which are each explored in the sections below.

Effects on photosynthesis

The exogenous application of most of the antioxidants reviewed here improves photosynthetic capacity in multiple crop species. Indeed, antioxidant-treated maize, wheat, cucumber, and melon (Cucumis melo) plants exhibit increased net CO2 assimilation rate, stomatal conductance, and transpiration compared to untreated plants (e.g., Yan et al. 2011; Li et al. 2016c; Wang et al. 2016a; Hasanuzzaman et al. 2017; Zhang et al. 2017c; Ali et al. 2019b; Huang et al. 2019). Generally, the benefit to photosynthesis from antioxidant application appears to be significantly higher during abiotic stress than under control conditions (Table 1, Fig. 2). For instance, foliar application of 200 μM melatonin to melon seedlings increased net CO2 assimilation by ~240% during cold stress and by only ~17% under control conditions compared to corresponding plants sprayed with water (Zhang et al. 2017c).

Experiments that assessed photosynthetic fluorescence parameters elucidated which specific biophysical improvements were imparted by antioxidant application (Fig. 2), including the maximal quantum yield of PSII photochemistry, the efficiency of excitation energy capture by open PSII centers, and the photosynthetic electron transport rate, which all increased in maize, tomato, melon, and eggplant (Solanum melongena) (e.g., Yan et al. 2011; Singh et al. 2015; Li et al. 2016b; Ding et al. 2017; Zhang et al. 2017c; Chen et al. 2018; Huang et al. 2019). Interestingly, the effect of antioxidant application on NPQ was not consistent across studies. For example, a tomato study reported an increase in NPQ following melatonin application (in combination with chilling, Ding et al. 2017), whereas two other studies of melatonin application to maize have shown a decrease in NPQ (Chen et al. 2018; Huang et al. 2019). Similarly, proline application has been shown to decrease NPQ (Singh et al. 2015; Noreen et al. 2018; Altuntas et al. 2020). Such discordant results suggest that exogenous antioxidants may interfere with redox states or the local proton gradients in a manner that perturbs NPQ formation differently in various species or growth conditions.

The benefits from applied antioxidants observed in multiple photosynthetic parameters are due to increased protection of photosynthetic enzymes from ROS-mediated damage. Multiple mechanisms have been proposed for such protection of photosynthetic enzymes, the simplest being that photosynthetic enzymes are directly protected by the capacity of the applied antioxidant to inactivate ROS. The most frequent mechanisms proposed tend to center on higher levels of native antioxidants and enhancement of antioxidant enzyme systems (e.g., Li et al. 2012b; Abdelhamid et al. 2013; Wei et al. 2015; Li et al. 2016c; Nourimand and Todd 2016; Odo 2016; Sadiq et al. 2016; Wang et al. 2016a; Hasanuzzaman et al. 2017; Pirbalouti et al. 2017; Zhang et al. 2017c; Irani and Todd 2018; Ali et al. 2019b; Yaqoob et al. 2019; El-Beltagi et al. 2020). Notably, direct scavengers of high-energy electrons in photosystems, chlorophylls, and carotenoids often accumulate to higher levels following exogenous antioxidant application (e.g., Yan et al. 2011; Li et al. 2012b; El-Sayed and El Sayed 2013; Alhasnawi et al. 2015; Wang et al. 2016a; Hasanuzzaman et al. 2017; Sadiq et al. 2019; El-Beltagi et al. 2020). Specific antioxidants, including proline and melatonin, have been linked to the upregulation of genes and the activity of their encoded enzymes relevant to light capture (e.g., chlorophyllase [Chlase], magnesium chelatase [Mg-CHLI], and multiple PSI and PSII components) and Calvin-Benson-Bassham cycle enzymes (e.g., phosphoenolpyruvate carboxylase [PEPc], Rubisco, and Rubisco activase) in maize and soybean (Wei et al. 2015; Altuntas et al. 2020), suggesting that antioxidants may increase photosynthetic capacity at the transcriptional and post-transcriptional levels (Fig. 2B).

The applied antioxidants may also interact with native antioxidant pools necessary for efficient photosynthesis. For example, applied glutathione may enter the cell and contribute to the highly reduced glutathione pool shown to be critical for high photosynthetic efficiency (Müller-Schüssele et al. 2020). Similarly, ascorbate is an electron donor for ROS production by PSI via the Mehler reaction (Asada 1999) and for an enzyme critical to NPQ (Bratt et al. 1995). Other antioxidants may act on photosynthesis through their effects on the ascorbate and glutathione pools, or independently. For instance, allantoin might serve as an alternative source of nitrogen during reduced photorespiration (Fahad et al. 2019). Thus, exogenous allantoin applications may alleviate the negative consequences of an increase in atmospheric CO2 levels on nitrate assimilation in leaves (Rachmilevitch et al. 2004). A similar phenomenon has been suggested for GABA, which also helps control the carbon-nitrogen balance (Bouche and Fromm 2004; Li et al. 2016a). Flavonoids are thought to protect membranes by promoting membrane remodeling to prevent oxidative damage (Erlejman et al. 2004). Possible mechanisms by which chlorophyll contents are affected include upregulating chlorophyll biosynthesis by alleviating the ROS-imposed inhibition of chlorophyll biosynthesis enzymes (Aarti et al. 2006) and limiting chlorophyll degradation via an upregulation of ferredoxin (Lin et al. 2013) or downregulation of chlorophyllase (Altuntas et al. 2020).

In summary, plants treated with antioxidants maintain a higher photosynthetic capacity during stress and frequently upregulate photosynthesis in control conditions. The general picture revealed by multiple studies shows that antioxidant-treated plants harvest more light energy and fix it, as measured by fluorescent parameters of photosynthesis and higher CO2 assimilation rates and faster growth (Fig. 2). A survey of studies in diverse plant systems and environments suggests that higher CO2 assimilation rates can be achieved in antioxidant-treated plants via a combination of an increase in the amount, stability, and activity of photosynthesis-related enzymes; an increase in the amount and stability of PSII and PSI; and remodeling of the photosynthetic membrane. The effect of antioxidants on NPQ is less clear and an interesting route for future study.

Effects on endogenous antioxidant systems

Plants have robust antioxidant systems composed of small molecular reductants and enzymes that assist these reductants in detoxifying ROS and regenerating reductive capacity. Among these molecular antioxidants that mitigate ROS levels are those covered in this review (Fig. 1) and free sugars (Couee et al. 2006; Foyer and Noctor 2011; Noctor et al. 2015). Of these, glutathione and ascorbate have central roles, as they connect the entire pool of antioxidant molecules to the reductive metabolism of the photosynthetic and respiratory electron transport chains (Fig. 3A, Noctor et al. 2018). Glutathione and ascorbate are also used as coenzymes for some antioxidant enzymes, and because they are of such central importance, they often accumulate in response to exogenous antioxidant application (Hasanuzzaman et al. 2019).

Antioxidant enzymes are very diverse in plants. Superoxide dismutase and catalase use metal cofactors to convert ROS into non-toxic forms. Ascorbate peroxidase, glutathione peroxidase, and glutathione S-transferase all detoxify ROS or other dangerous chemicals using ascorbate or glutathione as co-substrates. Finally, dehydroascorbate reductase, glutathione reductase, and monodehydroascorbate reductase regenerate reduced forms of glutathione and ascorbate from NAD(P)H or glutathione in the case of dehydroascorbate reductase. The presence and extent of endogenous antioxidant systems in plants have been the subject of several excellent reviews (Das and Roychoudhury 2014; Noctor et al. 2018), including those that cover the exogenous application of some antioxidants (Xu and Huang 2017; Hasanuzzaman et al. 2019).

Plant enzymatic and chemical antioxidants act synergistically to balance ROS production in multiple cellular compartments (Fig. 3A), as well as in the extracellular space: the apoplast (Fig. 3B). Higher expression levels of the genes encoding these endogenous antioxidative enzymes and, thus, higher activities have been reported to limit ROS damage and accumulation and improve plant tolerance to biotic and abiotic stresses (Foyer et al. 1995; Li et al. 2010; Dixit et al. 2011; Le Martret et al. 2011; Sharma et al. 2012; Diaz-Vivancos et al. 2013; Gaber 2014; Xu et al. 2014). Similarly, higher levels of reduced glutathione and ascorbate have been associated with increased tolerance to multiple stresses in several plant species (Foyer et al. 1991, 1995; Aono et al. 1995; Noctor et al. 1998; Lederer and Boger 2003; Hatano-Iwasaki and Ogawa 2012; Gill et al. 2013; Lisko et al. 2014; Cheng et al. 2015; Zhang et al. 2015b; Ali et al. 2019a; Xing et al. 2019; Bulley et al. 2021). When ROS production is not balanced, often because of an acute environmental change, damage imposed by ROS occurs along with ROS signaling. The extent of cellular injury is most frequently indirectly measured by the levels of a relatively long-lived lipid peroxidation product, malondialdehyde (MDA, Morales and Munné-Bosch 2019). Many of the studies above, in addition to detecting changes in the levels of glutathione, ascorbate, and antioxidant enzymes, also observed lower MDA levels upon application of antioxidants.

Given that the individual application of many antioxidants improves plant responses to stress conditions (Table 1) and that plant antioxidant molecules and enzymes contribute directly, it is not surprising that plants frequently accumulate increased levels of plant antioxidant molecules and enzymes when exposed to stress. For example, application of ascorbate increases antioxidant enzyme activity and reductant power in alfalfa (Medicago sativa), tomato, and wheat exposed to salt stress (Athar et al. 2009; El-Sayed and El Sayed 2013; Chen et al. 2021b); in maize exposed to cadmium stress (Zhang et al. 2019a); and in adzuki bean (Vigna angularis) and tall fescue (Festuca arundinacea v. Schreb.) exposed to waterlogging/hypoxia stress (Xu et al. 2015; Ullah et al. 2017). Cysteine applications increased the activity of catalase and superoxide dismutase during control and drought conditions in soybean (Teixeira et al. 2017). NAC application to black nightshade (Solanum nigrum) stressed with cadmium resulted in lower MDA levels and higher activity levels of antioxidant enzymes and the endogenous glutathione pool (Deng et al. 2010). Melatonin increased the activity of antioxidant enzymes when applied to chilled melon plants (Zhang et al. 2017c), excised apple leaves (Wang et al. 2012b), and osmotically stressed cucumber (Zhang et al. 2013) and grape (Vitis vinifera) plants (Meng et al. 2014). Application of α-tocopherol increased the levels of endogenous ascorbate in wheat plants challenged by salt or drought stress (Sakr and El-Metwally 2009; Ali et al. 2019b) and increased antioxidant enzyme activity in drought-challenged mung bean (Vigna radiata), soybean, and wheat (Sadiq et al. 2016; Hasanah et al. 2017; Ali et al. 2019b). These examples make it clear that antioxidant applications during stresses typically cause increased levels of endogenous antioxidants and antioxidant enzymes in multiple species.

Importantly, higher levels of endogenous antioxidants are not a universal property of antioxidant application, and there are clear indicators that high levels of antioxidants cause stress, rather than relieve it. When high levels of ascorbate were applied to Arabidopsis growing under normal conditions, plant growth and the activity of five antioxidant enzymes decreased, while MDA levels increased (Qian et al. 2014). Similarly, when NAC was applied to wheat subjected to heavy metal stress, endogenous glutathione and antioxidant enzyme levels decreased, depending on the metal and enzyme (Colak et al. 2019). These results might be interpretable within the context of ROS homeostasis. When homeostasis is disturbed by stress and ROS accumulate, the addition of an applied antioxidant should reduce ROS and restore ROS homeostasis. In contrast, high levels of antioxidants may cause stress by unbalancing ROS homeostasis. Future work exploring ROS homeostasis in the context of endogenous antioxidant additions could determine if such a simple explanation is correct.

Antioxidants applied to the outside of a plant, through foliar spraying, seed soaking, seed coating, or via watering for root uptake, will likely first interact with the apoplast, the extracellular space, before they reach the cytoplasm. To our knowledge, ascorbate is the only native antioxidant that has a function in the extracellular space (Fig. 3B, Hasanuzzaman et al. 2019). There, ascorbate levels are thought to help regulate developmental and signaling processes that depend on redox status (Podgórska et al. 2017) like plant responses to auxin (Pignocchi et al. 2006), SA signaling (Rahantaniaina et al. 2017), and cell wall loosening. Loosening of cell walls is a prerequisite for cell expansion and is linked to extracellular hydrogen peroxide levels (Lu et al. 2014). The rapid and transient accumulation of extracellular hydrogen peroxide also participates in the signaling mechanisms that plants deploy in response to both abrupt stresses like pathogen infections (Chaouch et al. 2012) and wounding and longer-term stresses like temperature changes, salt exposure, and drought (Miller et al. 2009). These processes are also likely to be influenced by apoplast ascorbate levels (Fig. 3B), which are maintained by a set of enzymes, including apoplast-localized ascorbate oxidase and cytoplasmic dehydroascorbate reductases (Xiao et al. 2021). These enzymes are the basis for the interconversion of apoplast ascorbate to oxidized apoplast ascorbate and oxidized cytoplasmic ascorbate to reduced cytoplasmic ascorbate. Apoplastic ascorbate is therefore connected with internal ascorbate levels and, thus, to the rest of the cellular reductants, most of which interact directly with ascorbate. Notably, the effects of disrupting apoplastic ascorbate are consistent with frequently observed effects of antioxidant application: bigger plants through increased cell expansion and response to auxin, more stress-tolerant plants through SA signaling, and changes to endogenous antioxidant levels. Apoplast-dependent effects would presumably be similar for all antioxidant applications that are reductants (see Fig. 1, glutathione, ascorbate, N-acetylcysteine, melatonin, tocopherol, thiamine, flavins, and chitosan), which may explain why different chemical antioxidants yield similar phenotypic consequences. Mechanistic details of the effects of exogenously applied antioxidants on apoplastic ascorbate are currently lacking but are worth exploring in the future.

Effects on phytohormones

Phytohormones help coordinate plant growth, development, and stress responses (Verma et al. 2016). The crosstalk between phytohormonal responses and their far-reaching effects makes them particularly powerful agents in reshaping the plant body. Additionally, native antioxidant levels are intrinsically tied to phytohormone production, as the antioxidant ascorbate is a cofactor for the production of GAs, ethylene, and ABA (Gallie 2013; Zhang 2013). Thus, the exogenous application of antioxidants frequently modifies phytohormone levels (Table 2). We separately consider the growth-promoting phytohormones auxin, GA, and cytokinin (Wang and Irving 2011; Verma et al. 2016) and the stress-related phytohormones ABA, SA, JA, and ethylene (Bari and Jones 2009). Note that while these phytohormones are grouped for simplicity, their individual roles reach beyond these simple “growth” or “stress” designations.

Table 2 Effects of exogenous antioxidant application on phytohormone levels
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Considering that most antioxidant applications lead to greater plant growth (Table 1), it is perhaps surprising that the levels of growth phytohormones are not consistently higher. Individual applications of glutathione, ascorbate, and GABA have been shown to raise auxin levels in Arabidopsis, maize, citrus (Citrus sinensis), and cucumber (Table 2; Koprivova et al. 2010; Dinler et al. 2014; Cheng et al. 2015; Hijaz et al. 2018; Guo et al. 2020), while applications of ascorbate and melatonin lowered their levels in Arabidopsis (Table 2; Wang et al. 2016b; Ren et al. 2019). Because plant hormones are involved in growth and development, some or all of their effects likely depend on the plant growth stage. Indeed, phytohormone changes appear to depend on growth stage in maize when ascorbate is applied and auxin levels are measured (Dinler et al. 2014). Few studies have measured either GA or cytokinin levels, which have been reported to increase in response to application of melatonin on cucumber (Zhang et al. 2014), proline on tobacco (Dobra et al. 2010), or ascorbate on wheat (Sadak et al. 2013).

While some data suggest that exogenous antioxidant applications support increased growth under normal conditions, the same treatments generally increase growth during stress conditions (Table 1). Likewise, exogenous antioxidant applications tend to similarly affect stress-related phytohormone levels. For instance, ABA levels increased in response to treatment of Arabidopsis, wheat, maize, citrus, poplar (Populus trichocarpa), and grape with the chemicals glutathione, ascorbate, GABA, allantoin, and melatonin (Table 1; Sadak et al. 2013; Dinler et al. 2014; Watanabe et al. 2014; Cheng et al. 2015; Hijaz et al. 2018; Ji et al. 2018; Xu et al. 2018). Indeed, activation of ABA was suggested as the major molecular mechanism by which increased stress tolerance was achieved in the case of allantoin application (Watanabe et al. 2014). Abiotic stresses are well known to cause spikes in ABA levels in plants and that pre-application of ABA improves abiotic stress tolerance in a variety of species (Olds et al. 2018). Similarly, SA levels increased in plants exposed to biotic or environmental stress (Hayat et al. 2010), and SA-based changes are thought to be how BTH affects plant tolerance to biotic stress. BTH is considered a functional analog of SA (Klessig et al. 2018). SA levels also increased in response to applications of glutathione, ascorbate, and GABA in Arabidopsis, maize, and citrus (Mhamdi et al. 2013; Dinler et al. 2014; Hijaz et al. 2018; Sultana and Chattopadhyay 2020). SA levels have been shown to increase when ascorbate levels are lowered genetically and to decrease when glutathione levels are genetically lowered (Brosché and Kangasjärvi 2012), suggesting that SA may be a sensitive indicator of cellular antioxidant status, possibly through H2O2 levels (Han et al. 2012). The levels of JA and ethylene are less frequently measured compared to SA and ABA. However, JA levels appeared to increase after application of glutathione, GABA, or allantoin in Arabidopsis and citrus (Mhamdi et al. 2013; Cheng et al. 2015; Takagi et al. 2016; Hijaz et al. 2018), and ethylene levels rose in response to GABA and melatonin in sunflower (Helianthus annuus), Caragana intermedia, poplar, and grape berries (Kathiresan et al. 1997; Shi et al. 2010; Ji et al. 2018; Xu et al. 2018). Increased levels of these stress-responsive phytohormones are not absolute. Again, ABA and SA levels depend on the plant species, growth stage, and time of sample collection relative to the time of application (Dinler et al. 2014). Despite these possible limitations, relatively few studies have reported any decrease in the levels of stress-related phytohormones in response to antioxidant application (Table 2). In summary, the analysis of phytohormone levels lends support to the observation that exogenously applied antioxidants are of greater benefit under stress conditions.

Challenges to agricultural use of exogenous antioxidant applications

In the sections above, nearly all the references pertain to the effects of applied antioxidants in a laboratory or greenhouse setting, and many of them point to the positive influences of antioxidants on plant growth or stress tolerance. It is attractive to translate these small-scale studies to improve plant resilience at the scale of production agriculture, and some work suggests this is a desired long-term outcome of the research. In the sections below, we briefly introduce the remaining major hurdles of using exogenous antioxidants in agricultural settings.

Transitioning antioxidant application from the laboratory to the field

The most common method used to evaluate laboratory applications of antioxidants is by addition of the compound to synthetic plant growth medium. For example, glutathione, ascorbic acid, melatonin, thiamine, and allantoin have all been tested in this manner (Ramirez et al. 2013; Cheng et al. 2015; Yang et al. 2021b; Tunc-Ozdemir et al. 2009; Irani and Todd 2018). The use of synthetic growth media is an advantage in isolating the impact of antioxidant treatment; however, its relevance to field conditions is nominal. A similarly irrelevant lab method to isolate the effect of an antioxidant is through direct infusion by inserting needles into the vasculature, for example, in soybean (Bashor and Dalton 1999). Agriculturally, infusions are used with a few high-value products, including grapevine and tree crops, but are not economical for most crops.

The two antioxidant application methods most likely to transition to agricultural field management strategies are foliar spraying and seed treatments. Foliar spraying allows a liquid to be applied directly to plant canopies and has been used to apply each antioxidant covered in this review (e.g. Chen et al. 2012; Li et al. 2016c; Abdelhamid et al. 2013; Ramirez et al. 2013). Foliar spraying is broadly compatible with available whole-field spraying mechanisms, e.g., pivot irrigation, herbicide, or pesticide application. Typically, lab strategies include frequent spraying early after plant emergence from the soil, while most irrigation strategies are used later during the hotter, drier months of late summer. Herbicides are frequently applied early in the season, and pesticides are applied later in the season. Combining these sprays with antioxidants may be a feasible mechanism for agricultural deployment if development of formulas effectively combining the herbicide or pesticide with an antioxidant is possible. Droplet size and spray distance may also need to be optimized for antioxidants as spraying a solution generates small droplets with large surface areas, encouraging oxidation. Seed soaking primes a seed for germination by simply soaking it in a solution containing antioxidants and has positively influenced germination (Xia et al. 2020) and early growth for multiple antioxidants (Burguieres et al. 2007; El-Beltagi et al. 2020). In an agricultural setting, seed soaking would not work for many crops as the soaked seeds cannot be used in automated planters. The agricultural equivalent is seed coating. Seeds can be coated with a liquid antioxidant slurry that is dried to a solid shell around the seed. Subsequent planting can be performed with automated planters. In fact, one study of coated soybean seeds shows that germination improved when chitosan was included (Zeng et al. 2012). As for foliar spraying, seed coating methods must be engineered to reduce oxidation of the antioxidant.

In summary, lab methods must be converted to field applications through a systematic engineering approach. There are likely to be differences in effectiveness between methods and between antioxidants based on their intrinsic chemical stability. For example, ascorbate application was more effective in mitigating salt stress in wheat when applied in the rooting medium compared to seed soaking or foliar spray (Athar et al. 2009). There are often unexpected conditions in field environments, such as highly variable light in the canopy and simultaneous combinations of abiotic and biotic stresses that make prediction of agricultural applications from lab tests challenging. However, there is evidence that some antioxidants can be effective in improving plant characteristics in field settings, including GABA, proline, tocopherol, thiamin, and chitosan (Table 1). These studies imply that the transition from lab methods to field management strategies is worth pursuing.

Effects on surrounding ecosystems

Antioxidants broadly support the health of plants and animals, and agricultural use of antioxidants may promote greater plant growth and productivity. However, large-scale agricultural application of antioxidants may also have ecological consequences, particularly when concentrations exceed what naturally occurs (Qian et al. 2014). To date, very few studies have evaluated the effects of antioxidant application on the surrounding environment. Here, we use available work to extrapolate possible consequences associated with antioxidant application for plants, microbes, and animals in terrestrial and aquatic ecosystems.

Antioxidant applications may result in different outcomes as a function of the plant species. For example, ascorbate application promoted growth in mung bean, tomato, and sunflower exposed to heat, salt, and osmotic stress, respectively (Shalata and Neumann 2001; Younis et al. 2010; Kumar et al. 2011), while in Arabidopsis, ascorbate application resulted in ROS overproduction and plant growth inhibition (Qian et al. 2014). Thus, an antioxidant application may inadvertently provide a further advantage to weedy or otherwise ecologically undesirable species.

Antioxidants applied to plants will certainly affect nearby microbes, which may in turn influence plant health (Lakshmanan et al. 2014). Plants form symbiotic relationships with beneficial microbes, particularly those that colonize plant surfaces (the phyllosphere, Lindow and Brandl 2003) and those that reside in the soil in close proximity (0.5–4 mm) of their roots (the rhizosphere, Kuzyakov and Razavi 2019). Plants also influence microbial communities through allelopathy, that is, the release of allelochemicals in plant surfaces and root exudates that alter microbial spore germination, growth, survival, and reproduction (Marilley et al. 1998; Hein et al. 2008; Cheng and Cheng 2015). Several of the antioxidants considered in this review have been described as allelochemicals and modulate the redox homeostasis of microbes. For example, allantoin is deposited by rice and appears to stimulate shifts in microbial populations while increasing microbial diversity (Wang et al. 2010). Proline is exuded by citrus roots under salt and heat stress conditions (Vives-Peris et al. 2017). Independent studies of proline applied to microbes show that proline application improves osmotic stress tolerance in the bacterium Salmonella enterica (Csonka 1981) and budding yeast (Saccharomyces cerevisiae) (Takagi et al. 1997). Further research could clarify how symbiotic and pathogenic signals between plants and microbes are affected by application of antioxidants.

Beyond the crop and its homeostasis with surrounding microbes, agricultural fields are part of an ecosystem including insects, other invertebrates, birds, mammals, and local waterways (Fig. 4). For herbivorous animals and microbes, we know that long-term activation of plant defenses is likely to cause a negative impact similar to that seen from pesticide application. Studies of antioxidants in freshwater and marine ecosystems have involved mesocosms, often aimed at the production of specific organisms. The effects of antioxidants on various trophic levels, from bacteria to fish, have been described as both positive (Zhang et al. 2006; Harder et al. 2018), and negative. For example, GABA induces settlement and metamorphosis of bivalve mollusks, two important stages in commercial culturing (García-Lavandeira et al. 2005). In contrast, chronic exposure to o-hydroxyhippuric acid (HDP), an SA derivative created after consumption, caused multiple negative health effects for two species of cladocerans, Daphnia longispina and D. magna (Marques et al. 2004). Overall, the addition of some antioxidants at lower concentrations may be beneficial, although the ecosystem balance in response to antioxidants has yet to be studied.

Fig. 4
figure 4

Potential sources of antioxidant contamination in aquatic and terrestrial ecosystems due to exogenous applications. Contamination of the air, soil, and water by antioxidants can include point- and non-point sources. Applications by spraying may drift away from target areas due to wind or evaporation of contaminated surface water. Soil contamination may occur when antioxidants bind to the soil in high concentrations. Water contamination may occur through underground seepage or leaching or surface water runoff from contaminated fields. Most antioxidant contamination occurs near the field where antioxidants are applied, but long-distance transport may be possible

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In summary, a primary detractor to agricultural use of antioxidants is our lack of understanding of their ramifications on the ecosystem. Severe consequences may negate the usefulness of specific antioxidants. Thus, an appreciation of the ecological impacts could drive the choice of the antioxidant to apply, the effective dose, and the application method.

Conclusions and future directions

Antioxidants can be applied to various plants through multiple mechanisms to confer greater stress resistance and improved growth. The application of diverse antioxidants is associated with several shared direct effects on native antioxidant chemical and enzyme systems, improved photosynthetic capacity or flexibility, modified phytohormone signaling, and metabolism of the applied antioxidant. While the metabolism of each antioxidant is unique and may individually contribute to the observed effects, antioxidants may be grouped to reflect their similar positive effects, which may better elucidate the underlying mechanisms for observed benefits (Table 1).

At this time, the mechanism of how each antioxidant leads to a positive growth outcome and whether multiple antioxidants truly employ a similar mechanism or only yield comparable outcomes are unclear. From the prevalence of foliar applications with positive growth impacts, we suggest in the “Effects on Endogenous Antioxidant Systems” section that the apoplast may be similarly affected by any application of a chemical reductant (Fig. 1, GSH, ascorbic acid, N-acetylcysteine, melatonin, tocopherol, thiamine, riboflavin, and chitosan). In the future, this may be explored through comparing applications of reducing antioxidants and antioxidants whose primary effect is on endogenous antioxidant systems (GABA, proline, and BTH). Relatively few studies have compared how multiple native antioxidants affect the same species (e.g., Sakr and El-Metwally 2009), and none have fully investigated the apoplast. A major difficulty in comparing multiple native antioxidants is the unknown effect exerted by the specific metabolic pathway of each antioxidant. Creative approaches are thus needed for deciphering nonspecific chemical effects from direct redox responses. One option would be to group antioxidants by their reduction potential (Fig. 1), comparing the effects of applying two different chemicals with similar redox values. Another possibility would be to use “cheminformatics” to engineer chemicals with a similar structure but with no redox potential. The use of near-native antioxidants may offer a means to map the specificity by which native antioxidants are recognized and used.

Other basic questions remain about how plants use applied antioxidants that, when answered, should inform application strategies. Some of the most important questions we identified are as follows: To what extent and at what rate are antioxidants internalized with each application strategy, and in which cellular compartment(s)? Is there a systematic effect on endogenous antioxidant systems? Do antioxidants have dedicated receptors, as with melatonin (Wei et al. 2018)? Answering these questions would help better define the sets of antioxidants that are most interchangeable. NPQ becomes more important in the variable light conditions of an agricultural field. Are the NPQ effects varying by antioxidant compound or experiment type? Photosynthesis rates, phytohormone levels, and native antioxidant metabolism are known to cycle diurnally (Dodd et al. 2014; Atamian and Harmer 2016; Zechmann 2017). Therefore, to what extent does the rhythmic metabolic state influence the processing of antioxidants applied at different times of the day? The answer to this question may indicate to what extent timing of application, rather than compound, influences outcomes. What happens to the antioxidant after application? Does it contribute to plant redox homeostasis and regenerate, or is it used and then degraded for energy or nutrients? What is the turnover rate in each tissue to which it is applied? Do tissues transport the antioxidants away from their site of application? Answers to these questions would influence best practices for determining the dose, frequency, and site of application of individual antioxidants, and the precise answers may change based on the stress plants are experiencing.

We also identified clear actions for translating the growth influence of antioxidant application to the scale of agricultural production. First, some pilot experiments have already established that results can translate from greenhouse to field studies (Table 1); however, engineering the application strategy is a likely prerequisite to large-scale studies and then agricultural use. Of particular importance are questions about the safe use of antioxidant applications at scale. Antioxidants are generally considered beneficial for human health at relatively high doses in food. However, few studies have investigated the breakdown products of applied antioxidants and their risks to the environment (Fig. 4). Typical studies performed for environmentally applied chemicals have largely not been performed for antioxidants (see Effects on Surrounding Ecosystems). Similarly, the effects of consuming a plant that has been treated with antioxidants are unclear. A combined genetic, transcriptional, metabolic, and phenomics systems-level application study of antioxidant groups in key plant species would help assuage consumer safety concerns. Paired field and waterway testing would also assist in clarifying if environmental safety is a concern.

In summary, when different antioxidant compounds are applied to plants, they can have similar effects on plant resilience, photosynthesis, phytohormones, and endogenous antioxidants. More studies are needed to understand this unexpected congruity. Furthermore, before the growth benefits observed from antioxidant application can be realized in widespread agricultural use, we need critical knowledge on the mechanisms by which antioxidants produce their effects and on the safety of widespread agricultural use.