Ozone (O₃) is a destructive pollutant with negative effects on human and ecosystem health that induces abiotic stress in plants – affecting photosynthetic carbon assimilation, stomatal conductance, and plant growth – that damages crops and decreases crop yield (Ainsworth et al., 2012).

In plants, O₃ enters through the stomata and is degraded into secondary reactive oxygen species (ROS), including H₂O₂, O₂^⋅–, and HO^⋅, in the apoplastic space (Vainonen and Kangasjärvi, 2015). High levels of ROS can lead to ROS stress that causes direct or indirect ROS-mediated damage on a variety of molecules including lipid peroxidation in cellular membranes, protein denaturation, carbohydrate oxidation, and pigment breakdown (Sharma et al., 2012). Even more, ROS stress can eventually lead to changes in gene expression and even cell death. However, ROS are generated in the metabolism of plant cells and therefore plants need systems that scavenge oxidizing species. Constitutive enzymatic and non-enzymatic systems in cells protect them from ROS-induced damage and are inducible under biotic and abiotic stress that triggers a rapid increase in ROS, including O₃ exposure (Sachdev et al., 2021). Enzymatic ROS-scavenging mechanisms include antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GP), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) (Gill and Tuteja, 2010).

Specific ROS also act as “second messengers” in signal transduction pathways that lie downstream initial event in cells. The mitogen-activated protein kinase (MAPK) cascade is one of the signaling pathways sensitive to the cell’s redox status. Therefore ROS generation is a common consequence of diverse cellular traumas that activate the MAPK cascade and studies in different plants have demonstrated that O₃ exposure activates some components of the MAPK cascade (Marcus et al., 2000; Liu et al., 2015; Liu and He, 2017).

Studies on the effect of O₃ on agricultural productivity showed an estimated 5–15% crop yield loss in the United States (Avnery et al., 2011) and a 5% in Europe (Mills et al., 2007). To alleviate the drop in crop productivity, the main defense strategy over the past four decades has been the application of chemicals, mainly synthetic antioxidants (fungicides, insecticides, herbicides, nematocides, growth regulators, antitranspirants, antioxidants from the rubber industry, etc.) (Didyk and Blum, 2010). These xenobiotic products have varied protection capacity with some producing ineffective or even detrimental side effects. The most effective product was found to be EDU (ethylene diurea– (N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N0 phenylurea), developed by the duPont Chemical company (Archambault et al., 2000) whose main mechanism of action is not yet clear. Currently, more effective alternatives are being sought, such as extracts of plant origin, which do not generate environmental toxicity.

Rice Bran is a by-product of rice (Oryza sativa) milling that retains most of the bioactive compounds present in rice grain. These bioactives include naturally occurring antioxidants, mainly γ-oryzanol, tocopherols, tocotrienols, and polyphenols. Besides that, rice bran is also a good source of protein and fat (Goufo and Trindade, 2014). However, the low bioavailability of its biopolymers and bioactive compounds due to its high insolubility is the main drawback when considering rice bran as a biostimulant. Using enzymatic technology, our group has developed a process that enables obtaining a stable, water-soluble rice bran enzymatic extract (RBEE) from rice bran. RBEE has shown a high content in bioactive compounds, particularly phytosterols, γ–oryzanol, and tocols, and an increased content in peptides and free amino acids (Santa-María et al., 2010).

According to its content in bioactive compounds, RBEE has shown both in vivo and in vitro functional properties such as antioxidant capacity (Santa-María et al., 2010), hypocholesterolemic activity (Revilla et al., 2009), and antiproliferative and inmunoactivatory abilities (Revilla et al., 2013). Beneficial effects found in cells and animal models, especially those derived from its antioxidant capacity, lead us to consider the use of RBEE as a biostimulant in agriculture.

This work aims to evaluate the biostimulant capacity of RBEE. To this end, we propose studying the role of RBEE in plant protection against abiotic stress mediated by ROS, specifically in O₃ exposure. We chose pepper plants (Capsicum annuum) since pepper is a vegetable crop of great agricultural and economic importance being the second most traded spice in the world. Heavy losses in pepper production are frequently caused by abiotic stresses including O₃ exposition, in fact Capsicum pepper cultivation is almost entirely located in regions where O₃ concentration is increasing to phytotoxic levels (Bortolin et al., 2016). For these reasons pepper plants are an interesting research target to evaluate the negative effects of O₃.

Pepper plants exposed to O₃ were treated with RBEE and antioxidant enzyme activities and specific functional parameters were analyzed. Treatment with RBEE improved physiological parameters, such as net photosynthetic rate and photosynthetic pigments content in plants exposed to with O₃. RBEE treatment also decreased the induction of antioxidant enzyme activities in leaves (CAT, SOD, GPX, and APX) due to O₃ exposure.

Additionally, to search whether MAPK cascade is involved in the protective role of RBEE against O₃-induced damage we analyzed the transcriptional expression of CaMPK6-1, CaMPK6-2, and CaMKK5 genes. The high expression of genes studied strongly support that RBEE protective capacity involved the MAPK signaling cascade.

Biostimulants have been defined as “a formulated product of biological origin that improves plant productivity as a consequence of the novel, or emergent properties of the complex of constituents, and not as a sole consequence of the presence of known essential plant nutrients, plant growth regulators, or plant protective compounds” (Yakhin et al., 2017). Present results show a new product, an enzymatic extract from rice bran that protects against O₃-induced damage. O₃ treatment induces a decrease in net photosynthetic rate, chlorophyll content, and DF in pepper leaves which was partially reversed after foliar treatment with RBEE (Figures 1A,B, 2A, respectively). Thus, we can propose that RBEE may exert a biostimulant effect on pepper plants mainly based on its bioactive compounds.

Rice bran enzymatic extract, the enzymatically obtained extract from rice bran, shows a notable total antioxidant capacity (Revilla et al., 2009) and has been evaluated as a protector against lipid and protein oxidation in rat brain homogenate (Parrado et al., 2003). Additionally, we tested the antioxidant capacity of RBEE in two cell models: keratinocyte monolayers and reconstructed human epidermis both irradiated with UVB, and found that RBEE decreased lipid peroxidation in both systems (Santa-María et al., 2010).

Rice bran enzymatic extract is rich in prominent bioactive molecules. Many components of the extract may contribute to its antioxidant capacity. The most abundant phytochemical is γ-oryzanol, which is a natural antioxidant composed of ferulic acid esters of sterols and triterpene alcohols with high scavenging activity of free radicals, mainly mediated by ferulic acid moiety (Lemus et al., 2014; Minatel et al., 2016; Massarolo et al., 2017; Zduńska et al., 2018). RBEE also contains tocols (both tocopherols and tocotrienols) that act as antioxidants due to their ability to donate phenolic hydrogens (electrons) to lipid radicals (Xu et al., 2001). Polyunsaturated fatty acids which act as radical scavenging agents are also present in RBEE, contributing to the antioxidant activity. So, other natural substances such as Rosa rubiginosa oil (Franco et al., 2007), grape seed oil (Bail et al., 2008), and soybean-germ oil (Chen et al., 2019) are used for cosmetic purposes due to their polyunsaturated fatty acid content, which acts as a radical scavenging agent.

Rice bran enzymatic extract is also rich in PHs. PHs exhibit different antioxidant and free radicals scavenging activities (Li et al., 2008), mainly conferred by some nitrogenous compounds contained therein such as glycine, betaine, and proline (du Jardin, 2015). Moreover, PHs have shown an ability to enhance antioxidant mechanisms in plants (Gurav and Jadhav, 2013). It is worth noting that PHs, mainly those resulting from the enzymatic hydrolysis of protein substrates into low molecular weight peptides and free amino acids, have shown multiple biostimulant capabilities. Protein hydrolysates are included within the biostimulant classification (du Jardin, 2015). The direct effects in plants include modulating N uptake and assimilation, acting on signaling pathways in the root, regulating enzymes involved in this process (du Jardin, 2015), possessing hormonal activity similar to auxin and gibberellin (Colla et al., 2014), and producing antioxidant activity (Li et al., 2008; Gurav and Jadhav, 2013). In addition, when applied to soils, PHs have shown indirect effects on plant growth and nutrition by increasing the availability of nutrients and their acquisition by the roots – enhancing the microbial activity and biomass of the soil, soil respiration, and their fertility in general (du Jardin, 2015; Rodríguez-Morgado et al., 2015).

It is worthing to note that mixture of these bioactive molecules, as is the case of RBEE, has been reported to greatly enhance the antioxidant activity of rice bran oil (Perez-Ternero et al., 2017).

According to the RBEE antioxidant capacity, we have selected an acute O₃ treatment as an abiotic stressor. O₃ is degraded in the apoplast into secondary ROS (Vainonen and Kangasjärvi, 2015) and high levels of ROS can lead to ROS stress that induces the enzymatic and non-enzymatic-systems that protect cells from ROS (Sachdev et al., 2021). Accordingly all the enzymatic activities assayed – CAT, GPX, APX, and SOD – were induced after O₃ treatment. Induction was significantly reversed by the foliar treatment with RBEE (Figure 3). The effect on enzymatic activities could be correlated with the antioxidant effect of RBEE, thus the lipid peroxidation induced by O₃ treatment was significantly alleviated by RBEE (Figure 4A). Consequently, the antioxidant effect is reflected in the OSI values (Figure 4B).

Unlike other enzymes assayed, a surprising result was the induction of APX after the application of RBEE in plants not treated with O₃ (Figure 3B). We can speculate that this APX induction may be due to a hormetic effect induced by RBEE, stimulating a cellular system that could be essential for plant defense under stress. At the physiological level, hormesis is an adaptive response of an organism to a low-level stress factor accompanied by over-compensation when homeostasis is interrupted (Mattson, 2008; Calabrese, 2009; Wiegant et al., 2012). Hormesis is the cellular response in plants that occurs after an initial exposure to low levels of biotic or abiotic stressors – such as herbicides, temperature, chemicals, and radiation – which predisposes them to stimulate cellular defense mechanisms at subsequent sources of stress (Agathokleous and Calabrese, 2020; Berry and López-Martínez, 2020). For example, low doses of some herbicides such as 2,4-dichlorophenoxyacetic acid, glyphosate, and paraquat have been shown to trigger auxin production and antioxidant defense. The biostimulant-induced hormesis response of beneficial organisms allows plants to tolerate stress (Ahkami et al., 2017) through activation of secondary metabolisms and gene expression to recover homeostasis (Vargas-Hernandez et al., 2017). Application of biostimulants at right time can therefore facilitate increased plant growth while combined application of multiple biostimulants can be effective in reducing environmental drastic impacts (Dong et al., 2020).

In plants, the mechanism of hormesis is still unknown. The most probable pathways for hormetic responses are the induction of ROS by mild stress which leads to the activation of antioxidant defenses, stress-signaling hormones, or adaptive growth responses (Poschenrieder et al., 2013). Thus, induction of low and sub-toxic concentrations of ROS by mild stressors, such as which occurs as a result of foliar application of RBEE, has shown the ability to develop a hormetic effect, activating antioxidative defense and adaptive responses. RBEE is rich in 18C-unsaturated fatty acids (UFAs): oleic (18:1), linoleic (18:2), and α-linolenic (18:3) acids (Table 2). Besides their roles as ingredients and modulators of cellular membranes, reserves of carbon and energy, stocks of extracellular barrier constituents (e.g., cutin and suberin), or precursors of various bioactive molecules (e.g., jasmonates and nitroalkenes), recent works have pointed the role of 18C-UFAs as regulators of stress signaling (He and Ding, 2020). Oleic acid has been implicated in plant immunity against pathogens (Mandal et al., 2012). Even more, linoleic acid may regulate plant defense through ROS production (Yaeno et al., 2004). In this context, we can assume that RBEE application may induce a mild increase in ROS due to UFAs content that underly the induction of APX activity.

Ascorbate peroxidase is an H₂O₂-scavenging enzyme and is indispensable for the protection of chloroplasts and other cell constituents from damage by H₂O₂ and hydroxyl radicals (OH.). APX has been identified in most higher plants and comprises a family of isoenzymes present in different plant cell compartments, including apoplast (Sofo et al., 2015). As compared to catalase, it is more vital in stress as it has a high affinity for hydrogen peroxide (Afzal et al., 2014). Accordingly, we have found that APX activity exceeds catalase activity 10 times (Figures 3A,B). After O₃ enters the leaves through the stomata, it rises to the apoplast where it is immediately degraded into secondary ROS. Detoxification of ROS in the apoplast can therefore be considered as an early line of defense against O₃ (Kangasjärvi et al., 2005; Castagna and Ranieri, 2009). APX uses ascorbate as its specific electron donor to reduce H₂O₂ to water. Ascorbate is believed to be the major redox buffer and ROS scavenger in the apoplast (Foyer and Noctor, 2009). Thus, we can speculate that pretreatment with RBEE induced ROS generation that slightly stimulates APX activity, probably at the apoplast, the gate of entrance, that facilitates posterior induction and consequently protection against O₃ exposure.

The APX result could be working in tandem with the decrease in chlorophyll content and DF found in the same group of plants (Figures 1B, 2A). This effect could be due to linoleic acid present in RBEE as it has been shown that linoleic acid decreased the chlorophyll concentration and photosynthetic efficiency in Cylindrospermopsis raciborskii (Xu et al., 2016).

Protein phosphorylation mediated by protein kinase cascades is one of the most important post-translational modifications that coordinate response in cells. One relevant protein-kinase based amplification cascade is the MAPK cascade. The MAPK cascade is a complex signaling pathway hierarchically organized at least three sequentially acting serine/threonine kinases – a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and finally, the MAPK itself – with each phosphorylating, and hence activating, the next kinase. In this cascade, MAPKs phosphorylate specific substrate proteins, such as transcription factors and enzymes, and subsequently trigger cellular responses and rapidly transform upstream signals into appropriate intracellular responses (Liu et al., 2015). MAPK cascade is involved in many aspects of plant physiology, including cell division (Komis et al., 2011), plant growth and development (Xu and Zhang, 2015), plant resistance to pathogens (Bigeard et al., 2015) and insect herbivores (Hettenhausen et al., 2015), and plant response to abiotic stresses (Liu, 2012; Smékalová et al., 2014). It has been described as an interplay between MAPK cascade and ROS; so exogenous application of H₂O₂ or O₃ activates components of MAPK cascades. On the other hand, manipulating MAPK cascades results in initiation of ROS responses (Šamajová et al., 2013). In this context, the present work analyses the role of the MAPK cascade in the protective effect of RBEE against O₃ damage.

The majority of MAPK and MAPKK members are constitutively expressed in pepper plants. In leaves, the transcript level of CaMPK1 is the highest, followed by CaMPK6-2, and CaMPK19-2. Among MAPKK genes, CaMKK5 exhibits the highest transcript levels. Under the challenges of heat shock, salt stress, or pathogen inoculation, most of the MAPKs and MAPKKs in the pepper genome were significantly transcriptionally modified (Liu et al., 2015). Accordingly, after O₃ exposure, we found induction of the studied MAPKs. It is worth noting the relevant role that MPK3/MPK6 play in ROS response and O₃ sensitivity. AtMPK3/AtMPK6 in A. thaliana (Ahlfors et al., 2004) and the orthologs SIPK/WIPK in tobacco (Marcus et al., 2000), have been found to regulate for O₃ sensitivity; in fact RNAi-mediated silencing of MPK6 renders the plant more sensitive to O₃ (Miles et al., 2005). In these lines we found increased CaMAPK6-1 and CaMAPK6-2 after pepper plants exposition to O₃. Moreover, induction of CaMKK5 may be related to CaMAPK6-1 and CaMAPK6-2. It has been described in Arabidopsis that MKK5 was involved in MPK3/MPK6 activation in response to O₃ exposure (Vainonen and Kangasjärvi, 2015) and interestingly in pepper CaMPKK5 interacts with CaMPK3, CaMPK6-1, and CaMPK6-2 (Liu et al., 2015).

But surprisingly, after O₃ exposure, RBEE treated leaves showed a huge expression of these MAPK genes, being higher than in the control plants under O₃ in CaMPK6-1, CaMPK6-2, and CaMKK5 genes (Figure 5). As previously indicated, the MAPK cascade is involved in many aspects of plant physiology including the defense response against stress. The presence of multiple genes’ family members in genomes of different plant species encoding for MAPKKKs, MAPKKs, and MAPKs, and the fact that one MAPK cascade may be associated with more than one upstream or downstream partner (Liu et al., 2015) made it difficult to elucidate specific mechanism mediated by MAPK modules. How ROS activates MAPK cascades still remains unclear. It is possible that plants not only use MAPK cascades to transduce ROS signaling to gene expression and sometimes cell death, but also initiate the negative feedback regulation by MAPK cascades to maintain ROS homeostasis. The different combinations of the three tiers of kinases, distribution, time point-dependent activation, strength, duration, and availability of substrates of MAPK cascades may determine the feed-forward or feed-back outcomes (Liu and He, 2017). However, our results unequivocally indicate that both the damage induced by O₃ and the activation of the protection systems against it induced by RBEE take place through the transcriptional induction of the MAPK cascade.

Rice bran enzymatic extract, an enzymatic extract of plant origin, reversed the O3-induced decrease in physiological parameters as net photosynthetic rate, chlorophyll content, and DF. Thus, the results of our study highlight the potential use of RBEE as an effective biostimulant plant protector against oxidative stress caused by O₃. Present results also point out that MAPK cascade is involved in both, O₃-induced damage and RBEE protection. However, more studies are needed to clarify how this kinase pathway is involved.

Thus, we contribute to the general efforts done in the last decade searching new non-chemical alternative products to protect crops against damages caused by environmental oxidative stress.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

SM-B, SN-T, ER, AC, and JP designed the study. SM-B, SN-T, and LM performed the research. SM-B, SN-T, PC, ER, AC, and JP analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.