Ozone, an allotrope of oxygen, forms in the troposphere through complex chemical reactions between nitrogen oxides and volatile organic compounds, triggered by sunlight. This process is particularly pronounced in urban and industrial areas, where increasing human activity leads to a steady rise in tropospheric ozone levels (Wilkinson et al., 2012). Recognized as a major air pollutant, tropospheric ozone has detrimental effects on plant growth and productivity (Betzelberger et al., 2012; Wilkinson et al., 2012). The extent of ozone impact on plants depends on its concentration and the duration of exposure. Long-term exposure to low ozone levels reduces photosynthesis, stunts growth, and accelerates aging, often without visible damage to plant tissues. In contrast, short-term exposure to high ozone concentrations—an event that can occur multiple times during the growing season in various regions—triggers cell death and the formation of visible lesions in ozone-sensitive plants (Agathokleous et al., 2023; Nowroz et al., 2023). The grapevine (Vitis vinifera, L.) are sensitive to ozone, and elevated ozone levels can impair their photosynthetic performance, growth, yield, and fruit quality (Fumagalli et al., 2019).

The introduction of new legislation on recycling and the sustainable use of underutilized local resources in many countries has highlighted the importance of the circular bioeconomy for the manufacturing of high-value products (Spekreijse et al., 2019). Wine is one of the most widely produced alcoholic beverages globally, with an approximate production of 259.9 million hL in 2022. Typical wastes and by-products from wineries include grape pomace (composed of skins and seeds, which represent about 60% of total winemaking by-products, and stems, which account for approximately 14%), as well as grape solids and fermentation (yeast) lees (26%). Each year, an estimated seven million tons of grape pomace and lees are generated worldwide (Bordiga et al., 2019).

In recent years, there is a growing interest in reusing these food wastes not only to reduce their environmental impact by the circular production of by-products but also to entail an economic benefit derived from the reuse of products with added value (Baroi et al., 2022; Dwyer et al., 2014). Given a circular economy approach, some of these wastes can be successfully “recycled”, reused, or recovered, improving both the economic and environmental. These by-products are typically used for animal feeding, composting, industrial biomass, or distillate production (Bordiga et al., 2015). However, grape pomace and lees are high added-value by-products due to its wide variety of compounds. The wine lees are a combination of yeasts, organic acids, insoluble carbohydrates, inorganic salts, lignin, proteins, phenolic compounds, and ethanol (Jara-Palacios, 2019). These compounds are susceptible of extraction or transformation and exploitation with the consequent economic benefit.

Biostimulants are currently regarded as key tools in sustainable agriculture because they enhance specific plant functions that ultimately support crop performance under suboptimal conditions. In functional terms, plant biostimulants are products applied in small amounts that modulate plant physiological and biochemical processes, thereby improving nutrient use efficiency, activating or priming defense pathways, and increasing tolerance to abiotic stresses while contributing to yield and quality improvements (Russo and Berlyn, 1991; Yakhin et al., 2017). Historically, before the term “biostimulant” became widespread, expressions such as “biogenic stimulators” or “biogenic stimulants” were used for compounds produced in plant tissues under non-lethal stress that stimulated vital physiological reactions (Yakhin et al., 2017). The early concept of “organic plant biostimulants” proposed by Russo and Berlyn already emphasized their non-nutritional nature and their ability to enhance productivity and stress resistance in low-input systems (Russo and Berlyn, 1991). In line with recent literature and the current EU Fertilising Products Regulation (European Union, 2019), as well as related regulatory frameworks in other regions, biostimulants are now primarily defined from their agronomical function rather than their composition, focusing on their capacity to improve nutrient use efficiency and to prime plant defense and stress-response pathways, which in turn supports higher resilience and better crop performance.

In our group, we have previously developed microbiological and enzymatic technologies that allows obtaining biostimulants from agro-industrial residues, both of plant origin such as okara (García-Martínez et al., 2010; Orts et al., 2019, 2024) or rice bran (Macias-Benitez et al., 2021), and of animal origin such as chicken feathers (Caballero et al., 2020; Rodríguez-Morgado et al., 2014) or even from sewage sludge (Caballero et al., 2022; Rodríguez-Morgado et al., 2015; Tejada et al., 2013).

Considering the relevance of the wine industry in Mediterranean countries such as Spain, Greece, and Italy, regions with high tropospheric ozone levels, there is an emerging interest in developing protection strategies against ozono-induced oxidative damage (Doche et al., 2014; Wang et al., 2024). Such strategies are particularly valuable when they avoid environmental toxicity, as is the case with plant-derived extracts. In this context, the enzymatic technology developed by our group offers a dual advantage: it converts viticulture wastes into biostimulants aligned with circular economy principles, while simultaneously providing a sustainable tool to enhance plant resilience against ozone stress. This study specifically evaluates the protective role of these biostimulants in mitigating ozone-induced oxidative damage, bridging waste valorization with agroecological protection strategies.

The grapevine plays a fundamental role in the cultural, economic, and ecological aspects of the Mediterranean environment (Moura et al., 2023). Globally, this crop holds great economic importance (Blanco-Ward et al., 2021), and wine production is a key activity that is an integral part of the cultural identity of many countries (Ascenso et al., 2021).

The ability of tropospheric ozone to damage vegetation is widely studied, manifesting in reduced photosynthesis, accelerated cellular aging, increased susceptibility to diseases, decreased growth, and reduced reproductive capacity in plants. In the specific case of Vitis vinifera, it has been observed that it exhibits intermediate sensitivity to ozone (Ascenso et al., 2021).

The present study demonstrates the protective effect of a biostimulant (LEE) derived from red wine lees against ozone-induced damage in grapevine plants. The analysis of LEE composition reveals that it is a potential source of active molecules such as proteins and phenols (Table 1). The use of proteases enables the release of peptides from proteins, converting them into their active form (Justus et al., 2019). The resulting protein hydrolysates exhibit various biological functions, with antioxidant activity being one of the first recognized (Zhu et al., 2022).

Due to these properties, are considered an innovative alternative for stimulating plant growth. Their foliar application can help plants cope with abiotic stress by enhancing their antioxidant capacity (Sitohy et al., 2020). LEE also provides essential amino acids (threonine, valine, isoleucine, and leucine) that plants cannot synthesize and must obtain from the biostimulant (Sun et al., 2024).

The high content of phenolic compounds in the biostimulant further highlights its potential as an effective tool for enhancing plant health, as these compounds play a key role in plant development and defense mechanisms. Their multifaceted action significantly contributes to plant resilience and adaptation to adverse environmental conditions. Moreover, phenolic compounds are involved in the regulation of various signaling pathways associated with stress responses, underscoring their essential function in coordinating plant defense systems (Saini et al., 2024). Additionally, they have other applications in bioremediation and as antioxidants in food additives (Sharma et al., 2019).

The ozone treatment significantly affects the physiological state of the plants, causing a decrease in photosynthetic parameters, including A_N, ETR, PhiPII, and delayed fluorescence. The biostimulant treatment completely reversed these effects (Figures 1A-C; Figures 2A-B). Therefore, we propose that LEE may exert a biostimulant effect on grapevine plants by improving their overall physiological state.

Ozone-induced damage in living organisms is primarily due to its high oxidizing power. This damage mainly results from the generation of reactive oxygen species (ROS), either through the spontaneous decomposition of O₃ upon entering the apoplastic space or through direct interactions with various cellular components. These processes trigger oxidative damage to biomolecules, potentially compromising crucial cellular functions (Agathokleous et al., 2015).

Accordingly, the protective effect of LEE in plants exposed to ozone can be largely attributed to the antioxidant capacity of the biostimulant, as demonstrated by the DPPH and ABTS values (Table 1), a particularly relevant aspect to highlight. Another notable characteristic of this product is its osmoprotective ability, which is due to the high amount of the amino acid proline present in the hydrolysate (Table 2). According to Raza et al. (2023), plants accumulate this amino acid in response to various abiotic stresses. In addition to helping maintain osmotic balance, proline preserves cell turgor and indirectly modulates the metabolism of reactive oxygen species (ROS).

Currently, the advancement of omics sciences, driven by the development of genome sequencing technologies and the reduction in their costs, has transformed the understanding and identification of metabolic pathways in plants (Owen et al., 2017). To evaluate the effect of ozone and the biostimulant on plants, a transcriptomic analysis was performed using the RNA-Seq technique, allowing us to analyze global gene expression changes with high precision. The RNA-Seq analysis reveals significant changes in gene expression in response to ozone and the biostimulant treatment.

Compared to control plants, those exposed to ozone exhibit inhibited 50S and 30S ribosomal proteins present in chloroplasts and mitochondria (Table 3). These findings suggest a substantial impairment in the synthesis of proteins essential for key cellular processes such as photosynthesis, respiration, and antioxidant defense, ultimately compromising plant cell health and viability. Consistently, previous studies conducted on Arabidopsis thaliana exposed to elevated ozone concentrations (≥300ppb) reported a significant downregulation of genes encoding chloroplast ribosomal subunits (e.g., tAPX and Cu/Zn SOD, both associated with antioxidant pathways) and mitochondrial components (e.g., Mn SOD), thereby corroborating our results (Li et al., 2006).

Genes related to oxidative stress are differentially expressed, including transcription factors associated with phytohormones, highlighting, for example the ethylene-responsive transcription factors. Ethylene production, which can occur in high amounts following ozone exposure, may lead to cell death; however, ethylene at low concentrations can induce defense genes in Arabidopsis (Tamaoki et al., 2003). Proteins containing the BHLH (Basic Helix-Loop-Helix) domain were also found, as well as MYB, WRKY, and NAC transcription factors. Additionally, there was an overexpression of proteins that function as defense and adaptation mechanisms to minimize damage to proteins and other cellular components (Asthir, 2015; Foyer and Noctor, 2005). These include post-translational modifications, protein turnover, and chaperones, as well as the overexpression of glutathione S-transferases, oxidoreductases. These results align with findings in tomato, wheat, and barley plants (Gallé et al., 2009; Rezaei et al., 2013), where high expression of these enzymes correlated with greater tolerance to abiotic stress. This acclimation could also be due to redox regulations exerted by thioredoxins and glutaredoxins, which were differentially expressed upon ozone application. These two oxidoreductases regulate signal transduction pathways associated with plant growth, defense, and productivity (Jiménez et al., 2024).

The overexpression of pathways involved in the synthesis of biotin and phenolic compounds observed when plants are exposed to ozone provides valuable insights. On one hand, the activation of phenolic pathways (such as phenylpropanoids, stilbenes, and flavonoids) leads to the production of secondary metabolites associated with antioxidant activity, pathogen defense, and environmental stress resistance, as well as the strengthening of the cell wall. On the other hand, biotin is essential for fatty acid metabolism, gluconeogenesis, and abiotic stress responses, acting as a protective molecule (Cataldo et al., 2022; Noronha et al., 2023). These effects have been observed with the use of Ascophyllum nodosum (a brown seaweed) extracts, which stimulate antioxidant enzymes and phenylpropanoid pathways (including resveratrol) in tomato, enhancing defense against pathogens such as Botrytis and Plasmopara (Kanojia et al., 2024). Additionally, in Vitis vinifera, biostimulants have been shown to increase the accumulation of phenolics, biotin, and defense-related enzymes, thereby improving, for instance, drought tolerance (Irani et al., 2021). These pathways are crucial for wine quality and human health (Reinisalo et al., 2015).

The application of the biostimulant in ozone-stressed plants demonstrates that this product could mitigate stress by reducing oxidative damage in these plants while also enhancing resilience, as the plant may be better prepared to defend itself. This is evidenced by the overexpression of flavonoid and phenylpropanoid biosynthesis pathways in the presence of LEE (Bulgakov et al., 2024; Dao et al., 2025).

Another important aspect is resource conservation for the plant, as there is lower energy expenditure on defense due to the reduced activation of emergency stress responses. As shown in Table 4, the FoldChange of stress-related genes is less overexpressed or even repressed. Additionally, differentially expressed genes were found which play fundamental roles in signal perception and transduction. Studies have shown that the overexpression of these genes in Vitis vinífera model plants improves drought tolerance, indicating their potential for genetic improvement of crops under adverse conditions (Liu et al., 2024).

Therefore, the biostimulant alleviates the impact of ozone, optimizing metabolic efficiency and plant health.

In this study, we have analyzed the protective effect of a biostimulant obtained from red wine lees against abiotic stress induced by ozone in Vitis vinifera plants. Ozone has negative effects on both the growth and overall health of grapevines, which can lead to a decrease in productivity and wine quality. This is particularly concerning in areas where ozone concentrations are high due to atmospheric pollution.

The application of LEE effectively reversed the negative effects of ozone on plant physiology, improving photosynthetic activity and overall plant health. The antioxidant and osmoprotective properties of the biostimulant, along with its ability to influence key metabolic pathways, provide significant resilience against oxidative stress. In addition, the biostimulant optimizes resource use in plants by reducing the activation of emergency stress responses, thus supporting their growth and productivity. These results indicate that LEE may represent a valuable strategy to obtain a functional biostimulant from viticulture by-products, contributing to the circular use of grape-derived residues.

A limitation of this study is that all experiments were conducted under controlled greenhouse conditions on potted Vitis vinifera cv. Syrah plants, rather than in commercial vineyards. Consequently, the responses observed here may not fully represent the performance of grapevines grown under open-field conditions, and further trials in commercial vineyards will be necessary to validate the effectiveness and agronomic relevance of this biostimulant.