Bell pepper (Capsicum annuum L.) is an economically important vegetable crop which has a recent worldwide popularity among consumers in human diet due to its nutritional value, as an excellent source of biologically active compounds (vitamins, carotenoids, flavonoids, phenolic acids and other phytochemicals) with health-related properties, the crispness, and the versatility to be consumed as a fresh vegetable in salads, cooked meals or dehydrated for spices (Marín et al., 2004; Navarro et al., 2006; Serrano et al., 2010; Raybaudi-Massilia et al., 2017; Soare et al., 2017; Ge et al., 2020). Consumers have become more critical in the last decade with their purchasing decisions which are commonly focused not only in physical and sensory traits, such as color, size, pericarp thickness, firmness and flavor, but also in nutritional and nutraceutical characteristics (Rodríguez-Burruezo et al., 2010; Jiménez-García et al., 2018). In Spain, the total production of chili peppers and peppers (Capsicum annuum L. and Piper nigrum L, respectively) has steadily increased to more than 1.58-fold over the past 10 years, reaching over 1.5 million tons in the 2022 season (FAOSTAT, 2024). The primary postharvest challenges that result in substantial quality deterioration and diminished acceptability of bell peppers are as follows: Water loss, which leads to significant softening and shrinkage due to turgor pressure loss, starch degradation, and chemical modifications in the cell wall related to pectin by the action of softening enzymes, such as polygalacturonase (PG), pectin methyl esterase (PME), cellulase, and ß-galactosidase (Li Z. et al., 2024). On the other hand, peppers can experience chilling injury (CI) when stored at temperatures below 7-10°C. This leads to symptoms such as surface pitting, watery stains, browning of the seed and calyx, and fruit decay. This, in turn, can result in reduced marketability (El-Ramady et al., 2015; Charoenphun et al., 2024). Finally, there are pathological disorders, for example grey mold, which is mainly caused by Botrytis cinerea (Cheema et al., 2018).

Nowadays, global demand for high-quality vegetable products is rapidly increasing. However, climate change is negatively affecting agricultural areas and water resources which are decreasing (Sobczak et al., 2023). Bell pepper crop is sensitive to temperature fluctuations during the developmental and growth cycle, showing a lack of tolerance to high temperature since its fruit setting is drastically reduced when the day temperature rises above 32°C and/or the night temperature is above 20°C (Erickson and Markhart, 2002). This abiotic stress in the plant induces the production and accumulation of reactive oxygen species (ROS) leading to membrane breakdown and cellular turgor loss that can prevent plant growth and development (Preet et al., 2023). The process of scavenging ROS in plants as part of the antioxidant defense system and osmoprotectants comprises various antioxidant enzymes, including superoxide dismutase (SOD), which converts free superoxide (O₂) radicals to hydrogen peroxide (H₂O₂) and oxygen, ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD). The latter enzymes have shown to play a pivotal role in the detoxification of H₂O₂ through its decomposition into water and oxygen. Furthermore, the role of radical scavenging metabolites in ROS scavenging mechanisms is also of significance. In this sense, different phytohormones can positively influence the crop yield and reduce negative environmental impacts. On the other hand, fruit senescence and postharvest disease infection can result in quality and economic losses (Zhang and Jiang, 2019). Some plant hormones have been widely studied to improve the postharvest quality of fruit and vegetables (Zhang et al., 2020).

As the major endogenous component in signal transduction systems, SA plays an efficient role in plant growth and development, flowering, and fruit ripening, as well as in regulating photosynthesis (Natasha et al., 2020; Hadjipieri et al., 2021). On the other hand, SA is crucial in stimulating the systemic acquired resistance (SAR) in plants by regulating numerous biochemical and physiological functions related to tolerance to both biotic and abiotic stresses and modifying the antioxidant system (Khan et al., 2015). Therefore, SA and its derivatives elicit a wide range of metabolic and physiological processes in plants, which have great potential in reducing postharvest losses in horticultural crops. SA leads to the synthesis of proteins affecting several metabolic processes by regulating their gene expression (Janda et al., 2020). The mechanisms by which SA generates these improvements could be related to the protection of cell membranes, the increase in carbon metabolism, and antioxidant system, and the regulation of stress defense proteins (Kang et al., 2012; Sharma et al., 2017). However, its specific action mechanism is still not well understood. Indeed, different studies have demonstrated the hormonal interactions between SA and jasmonic acid (JA) and several different stress-link compounds taking place under abiotic stresses, highlighting the complexity of hormonal signaling cascades (Dempsey and Klessig, 2017; Devireddy et al., 2021; Huntenburg et al., 2022). Furthermore, the attenuating effects of SA in plants depend on the concentration used, the method of application, and the plant development stage (Nóbrega et al., 2020).

In recent years, the foliar application of exogenous SA to crops has shown to be effective in the regulation of biotic and abiotic stresses, increasing the yield of green pepper fruit by reducing stress-induced growth inhibition as well as fruit quality traits (Elwan and El-Hamahmy, 2009; Jiménez-García et al., 2018; Ibrahim et al., 2019; Veloso et al., 2021; Dobón-Suárez et al., 2021b; Ghahremani et al., 2023; Sobczak et al., 2023; Preet et al., 2023; Rodrigues da Silva et al., 2023). In addition, SA treatment was found to alleviate chilling injury in pepper fruits through enhancing antioxidant metabolism, fatty-acid desaturation efficiency and water retention (Fung et al., 2004; Ge et al., 2020; Hanaei et al., 2022). However, appropriate concentrations and methods of elicitor application need to be determined to improve the effectiveness of this practice under different growing conditions (Munshi et al., 2020; Sobczak et al., 2023). A recent review concluded that preharvest spraying provides better results than postharvest treatments, but the specific results need deeper research to be conducted focusing on the effect of spraying frequency time and growing environment on postharvest storage quality of fruit (Chen S. et al., 2023). Furthermore, Chen S. et al. (2023) stated that there is a lack of information about the metabolic mechanism of exogenous treatments with SA and its derivatives affecting fruit quality parameters, which is an emerging field that needs to be explored. In fact, most of the metabolomic and transcriptomic studies of SA available are related to the functions, biosynthesis or transcriptional regulations of this plant hormone for establishing resistance to many pathogens in plants (Ding and Ding, 2020). For instance, exogenous SA application bolstered resistance to Colletotrichum viniferum (Lin et al., 2024), Ralstonia solanacearum (Li N. et al., 2024), Podosphaera pannosa (Yang et al., 2022), Xanthomonas campestris pv. campestris (Sun et al., 2022), Colletotrichum (Shi et al., 2019), cucumber green mottle mosaic virus (Liu et al., 2023), and Penicillium expansum (Zhang et al., 2024) in grapes, tomato, roses, cabbage, tea, watermelon, and apples, respectively. Recently, Perez-Aranda et al. (2024) demonstrated that the expression of the CaPR1 (Pathogenesis-related protein 1) in Capsicum annuum seedlings, a marker gene employed as indicator of SA pathways activation, is down-regulated with SA elicitation (1, 2.5 and 5 mM) and the cross-talk between jasmonic acid/ethylene and SA mediated signal pathways for the regulation of this gene. Other studies have revealed that the transcriptional activation of SA signaling pathway, rather than its biosynthesis, plays a crucial role in the chilling and freezing tolerance of cucumber and potato fruit, respectively (Sim et al., 2024; Chen L. et al., 2023). However, Nguyen et al. (2024) reported that the ripening quality of mango mirrored the induced SA and jasmonic acid (JA) endogenous levels after liquid methyl salicylate (MeSA) fumigants in postharvest and correlated with the high expression of biosynthetic-related genes. In this sense, in-depth study of both foliar spraying and irrigation methods to pepper plants has received little scientific attention, and the systematic investigation of the mechanism and molecular bases of SA effects in the postharvest physiological process of green pepper fruit remains unclear, which restricts the possibilities for improvement of green pepper fruit quality using preharvest elicitation tools. Therefore, this paper aims to compare the effect of SA preharvest treatment applied by foliar spraying or irrigation on ripening and senescence of green pepper fruit during postharvest storage from an antioxidant metabolism approach to provide deep insight into a practical application of SA on extending the storage shelf-life.

Bell pepper (Capsicum annuum L.) is a model for studying the ripening and senescence processes of non-climacteric fleshy fruit, during which numerous physiological changes occur, the most noticeable being the color change caused by chlorophyll degradation and the synthesis of new pigments such as carotenoids (Chaki et al., 2015; Ma et al., 2021). Nevertheless, numerous changes occur during the ripening of bell pepper fruit, including alterations in flavor, aroma, and texture, which are regulated by both external and internal factors (Palma et al., 2011; Klie et al., 2014). For instance, phenological stages and harvest dates are two key factors that significantly influence some nutritional and functional traits of green pepper fruit, as it was unveiled by Dobón-Suárez et al. (2021b). Fruit ripening and senescence involve complex and highly coordinated molecular and biochemical processes that include ripening-associated genes, transcription factors, enzymes, repressors, signaling molecules, and metabolic pathways in both climacteric and non-climacteric fruits (Cherian et al., 2014; Fuentes et al., 2019). These processes influence fruit quality on one hand and postharvest losses on the other.

As fruit ripens or undergoes senescence, it becomes more susceptible to fungal pathogens (Alkan and Fortes, 2015), leading to green pepper fruit deterioration. Cold storage is widely adopted to prevent premature ripening and senescence since this fruit is highly perishable at ambient temperatures. However, green bell pepper is susceptible to chilling injury (CI) at temperatures below 7°C (Lim et al., 2007). Consequently, common strategies to slow down senescence and preserve fruit quality include both pre- and postharvest management practices and technological tools. Many factors, however, such as various plant hormones and biotic and abiotic stresses are known to influence bell pepper fruit ripening (Sun et al., 2015; Cheng et al., 2016). Recently, numerous studies have shown that salicylic acid (SA) preharvest treatment applied by foliar spraying influences the ripening and senescence of fruit species, such as sweet cherry (Giménez et al., 2014), table grape (Champa et al., 2014; Gomes et al., 2021), jujube fruit (Shanbehpour et al., 2020), pomegranate fruit (García-Pastor et al., 2020), lemon (Serna-Escolano et al., 2021) and green pepper (Dobón-Suárez et al., 2021a), showing activation of the antioxidant system and a delay in fruit senescence, as it was highlighted by Chen S. et al. (2023). The present study showed that SA preharvest treatment applied by both foliar spraying and irrigation enhanced the fruit quality of green pepper fruit resulting in a slowdown of the ripening and senescence processes during postharvest storage at 7°C ( Supplementary Figure 5 ). As it can be observed in Figure 7 , this fact could be modulated throughout the stimulation of the antioxidant system accompanied by an increase on the content of secondary metabolites and the activity of antioxidant enzymes that is mediated by the upregulation of their codifying gene expressions.

Fruit quality is related to the ripening and senescence of fruit during storage, which is involved in fruit softening, weight loss, color change, ethylene production and respiration rate (Gao et al., 2020). Salicylic acid (SA) regulates growth in plants, playing an efficient role in growth and development, flowering and fruit ripening, and photosynthesis (Natasha et al., 2020). Many studies have also indicated that SA and its derivatives play an important role in regulating the physiological metabolism of fruit to achieve optimal fruit quality and to maintain it during postharvest (Valverde et al., 2015; Giménez et al., 2016; Hanif et al., 2020; Amiri et al., 2021; Chen S. et al., 2023). Over the past ten years, biochemical data have also indicated that the bell pepper fruit ripening process is influenced by the metabolism of reactive oxygen species (ROS) and nitrogen oxygen species (NOS) (Palma et al., 2015; Corpas and Palma, 2018; Corpas et al., 2018), which reflects the profound biochemical and molecular changes taking place during ripening (Camejo et al., 2015). The accumulation of the reactive oxygen species (ROS) including hydroxyl radical, superoxide and hydrogen peroxide during fruit ripening can cause oxidative damage leading to membrane lipid breakdown and loss of cellular turgor, triggering cell death and damage in fruit tissue (Champa et al., 2014). The beneficial effect of SA has been recently related to its capacity to improve photosynthesis and the activity of antioxidant enzymes, leading to maintaining the balance between the production and elimination of ROS (Batista et al., 2019). In the present study, results show a significant effect of both foliar and irrigation SA on activating the APX and POD by upregulating the relative CaAPX and CaPOD gene expression, respectively, at harvest ( Figures 5A, C , 6A, C ). Results showed that SA treatment applied by foliar spraying was more effective on increasing CaPOD gene expression than SA, irrigation treatment, although this effect was not reflected in a higher POD activity in those foliar SA-treated peppers than the irrigated ones ( Figure 5C ). Nevertheless, SA treatment applied by irrigation was the most effective in stimulating CAT activity at harvest ( Figure 5B ), although this effect was not mediated through the upregulation of the relative CaCAT gene expression ( Figure 6B ). The effect of SA treatment on activating the antioxidant enzymes activity at harvest has been demonstrated in the present study and results show that this plant growth regulator could have a potential effect modulating the antioxidant enzymes-gene expression. These results propose that the enzymatic antioxidants can offset the damaging effects of ROS metabolism on cell structure (Gomes et al., 2021; Serna-Escolano et al., 2021), which results in a slowdown in ripening process and an increase on quality traits at harvest ( Figure 1 ). In fact, the ripening index (RI) was deeply correlated (negatively, Pearson) with firmness, color, TSS, TA, chlorophylls, phenolics, antioxidant capacity and antioxidant enzymatic system, except with the carotenoids content which also showed a similar correlation pattern ( Figure 8A ). Some studies corroborate these findings regarding the activation of enzymatic systems by the application of SA and its derivatives [Methyl salicylate (MeSA) and Acetylsalicylic acid (ASA)], leading to the high antioxidant activity in sweet cherry (Giménez et al., 2014; Zhang et al., 2011), pomegranate fruit (García-Pastor et al., 2020), citrus (Zhu et al., 2016), papaya (Hanif et al., 2020) and banana (Xu et al., 2019).

Phenolic compounds have antioxidant activity, which not only scavenge free radicals and reduce oxidative damage to fruits, but also contribute to fruit flavor and quality maintenance. Their biosynthesis is involved in phenylpropanoid pathway of plant secondary metabolites. Phenylalanine ammonia-lyase (PAL) is the first step to catalyze the conversion of phenylalanine to cinnamic acid which is further converted to phenolic acid (Oraei et al., 2019). The enzyme activity of PAL could be enhanced by SA (Zhou et al., 2018), increasing phenolic content in oranges (Amiri et al., 2021) and table grapes (Blanch et al., 2020). In the present study, SA treatment applied by foliar spraying upregulated the relative CaPAL gene expression at harvest ( Figure 6D ), which led to a significant increase in the total phenolic content ( Figure 3A ). However, the irrigation-SA treatment also showed an enhancement in phenolic compounds as compared with control ( Figure 3A ), although these findings were not corroborated by the analysis of the CaPAL targeted gene ( Figure 6D ). In this sense, previous transcriptomic and metabolic profiling of watermelon uncovered the role of SA pretreatment up-regulating the expression of flavonoid biosynthesis genes, thus increasing the total flavonoid content (Liu et al., 2023). Furthermore, transcriptome analysis and exogenous SA treatment demonstrated that SA (NPR1) is involved in the positive regulation of flavonoid biosynthesis (Wu et al., 2021).

On the other hand, SA treatment increased ascorbic acid content which is an essential plant antioxidant vital for defense against oxidative stress, leading to alleviating damages induced by ROS accumulation. In this sense, the content of both AA and DHA was quantified at harvest ( Figures 3B, C ), as well as the relative CaDHAR2 gene expression which is involved in the biosynthesis of the cytoplasmic enzyme DHAR2, namely dehydroascorbate reductase, implicated on the catalyzation of the glutathione (GSH)-dependent reduction of dehydroascorbate and had a direct role in regenerating ascorbic acid ( Figure 6E ). Results suggest that both SA applications (foliar and irrigation) effectively enhanced the AA and DHA content, and consequently, the total vitamin C content, throughout the upregulation of the CaDHAR2 gene expression at harvest, although no significant differences were appreciated between both methods ( Figures 6E , 3B, C ; Supplementary Figure 3A ). The form of AA can neutralize radicals to retard oxidative reactions triggering the ripening process in plant tissues (Huang et al., 2017; Sangprayoon et al., 2019). Other studies corroborate these findings hypothesizing that the application of SA could enhance the antioxidant ability by inhibiting the ascorbic acid oxidase (AAO) enzyme, which would affect the ascorbate-glutathione cycle positively, leading to the high content of AA in orange fruit (Amiri et al., 2021; Hanif et al., 2020; Wei et al., 2011). Other reports indicated that SA, MeSA, or ASA treatments could lead to high content of DHA in fruit, which is oxidized from AA by the AAO enzyme in pomegranate fruit and table grapes (García-Pastor et al., 2020; Hazarika and Marak, 2022).

Bell pepper ripening is characterized by important visual and metabolic changes regulated by transcription factors, with color changes caused by chlorophyll degradation and biosynthesis of new pigments such as carotenoids (Chaki et al., 2015). In the present study, both SA treatments applied by foliar spraying and irrigation significantly influenced the content of these pigments at harvest ( Figures 2A, C ; Supplementary Figure 2A ), the highest levels of chlorophylls a and b were recorded in those green pepper fruits harvested from 0.5 mM SA-treated plants ( Supplementary Figure 5 ). Multiple biological functions have been reported for chlorophylls as lipophilic-nature pigments. Strictly related to their antioxidant capabilities, two main mechanisms can be described: Their direct free-radical-scavenging activity and the metabolic activation of detoxification pathways, as was reported by Pérez-Gálvez et al. (2020). Accordingly, exogenous SA increased chlorophyll content under drought and salinity conditions (Tang et al., 2017; Ghassemi-Golezani et al., 2018). Some studies showed that there was a positive correlation between TPC and TAA (Amiri et al., 2021; Wei et al., 2011), considering also the contribution of ascorbic acid. The present study shows the positive correlation observed between total phenolics and AA and DHA content ( Figure 8A ). In this sense, the H-TAA ( Figure 4A ) was significantly improved by SA preharvest treatments at harvest, although the highest stimulation was achieved with foliar spraying. Since no significant differences were recorded between the two application methodologies on total phenolics or vitamin C content ( Figures 3A–C ; Supplementary Figure 3A ), probably other hydrophilic antioxidant compounds, such as glutathione (GSH), could be highly influenced by foliar SA treatment. As opposed to H-TAA, the analyses of the L-TAA at harvest showed that this parameter was stimulated by both SA applications, although no significant differences were observed between both methodologies ( Figure 4C ). This functional increment with the application of SA at 0.5 mM is corroborated by a previous study in ‘Lamuyo’ green pepper fruit and could be related to the enhancement chlorophyll a and b content by the application of SA that has been demonstrated in the present study ( Figures 2A, C ; Supplementary Figures 2A , 5 ). Preharvest applications of SA have been reported to increase the antioxidant capacities of grapes (Champa et al., 2014; Gomes et al., 2021), Indian jujube (Shanbehpour et al., 2020), lemon (Serna-Escolano et al., 2021), pomegranate fruit (García-Pastor et al., 2020), and sweet cherry (Giménez et al., 2014). Accordingly, SA preharvest treatment has a beneficial impact on the quality of green pepper fruit, ‘Herminio’ cv., at harvest which results in a slowdown in the ripening process ( Figure 1 ; Supplementary Figure 5 ). At harvest, quality traits such as color and firmness were positively correlated and both showed a deep correlation (positively, Pearson) with the antioxidant compounds and the activity of the antioxidant enzymes, although no correlation was observed with the targeted genes ( Figure 8A ). Specifically, the relative expression of CaDHAR2 gene was highly correlated with firmness, color, chlorophylls, phenolics, AA, DHA, L-TAA, APX and the expression of CaAPX gene ( Figure 8A ; positively, Pearson). The action mechanism proposed in the present study is the following: 1) SA induces the synthesis of secondary metabolites and enhances the antioxidant systems by stimulating the phenylpropanoid biosynthesis pathway, and 2) SA activates the antioxidant enzymes, acting on the modulation of the relative antioxidant systems-based genes expression and regulating the balance between the antioxidant system and ROS metabolism, contributing to antioxidant metabolism insights ( Figure 7 ).

The senescence delay triggered by the foliar and irrigation application of 0.5 mM SA extended the shelf-life of ‘Lamuyo’ green pepper fruit since the preharvest treatment reduced fruit quality losses ( Figure 1 ; Supplementary Figure 5 ). The contribution to extending shelf-life and delaying fruit senescence for 28 days at 7°C could be attributed to the enhancement of both total phenolics ( Figure 3D ) and antioxidant capacity from hydrophilic and lipophilic phases ( Figures 4B, D ) with both SA applications which might reduce the oxidative damage ( Figure 7 ). Regarding the functional increment related to phenolics content after harvest, results showed the highest increase rate in those green pepper harvested from SA-treated plants. However, the highest increment on H-TAA after 28 storage days was observed in those pepper fruits irrigated with SA, leading to a higher stimulating effect of the antioxidant system in those pepper fruits treated with SA. Similar findings applying salicylates in preharvest have been obtained in sweet cherry (Valverde et al., 2015; Zhang et al., 2011), pomegranate fruit (García-Pastor et al., 2020), lemon (Serna-Escolano et al., 2021) and plum (Davarynejad et al., 2015). Other contributing aspects to the delay in senescence could be related to the higher levels of two forms of ascorbic acids (AA and DHA; Figures 3E, F ) quantified in those green pepper fruits treated with foliar and irrigation SA. Moreover, foliar and irrigation SA treatments lead to higher increase rate on DHA and AA content, respectively, compared to control from harvest until 28 days of storage. In this sense, Ruoyi et al. (2005) indicated that this effect is mediated by the inactivation of the AAO enzymatic activity, which might have caused a delay in the senescence of peppers in the later stages of the storage period. The inhibition of AAO was also advantageous in keeping vitamin C and for anti-browning in sweet pepper (Rao et al., 2011). Recently, the coordinated regulatory network of ncRNAs involved in the ripening of bell pepper fruit has been analyzed (Zuo et al., 2019), providing a theoretical basis for deciphering novel mechanisms of fruit ripening in future studies. In the present study, the increase in secondary metabolites, such as phenolics or vitamin C, mediated by SA was not correlated with the upregulation of the relative CaPAL gene expression after 28 days of storage at 7°C, as it can be observed in Figure 8B . Similarly, both SA applications did not modulate the response of CaDHAR2 gene after 28 storage days ( Figure 6J ). This finding could be related to the fact that SA was applied in preharvest upregulating both genes at harvest, and probably during the crop cycle, although the modulation effect is lost during postharvest storage.

On the other hand, a delay in senescence is commonly associated with a delay in color changes and this effect was observed in the present study from a metabolomic approach, where chlorophylls a and b content were significantly higher in both foliar and irrigation SA-treated pepper fruits than control ( Figures 2B, D ; Supplementary Figure 2B ), as it was previously discussed. In addition, the rate of decline on chlorophyll a and b from harvest date until the end of postharvest storage was lower in both SA treatments, especially with the irrigation method, compared to control. This result shows a preserving effect on green color maintenance after postharvest storage associated with the SA treatments, as it can be observed in Supplementary Figure 5 . After 28 days of storage at 7°C, a similar effect was observed on the relative expression of CaAPX and CaCAT genes which was no upregulated by SA treatments ( Figures 6F, G ), although an enzymatic activity stimulation was observed compared to untreated pepper fruit because of both foliar and irrigation SA treatments ( Figures 5D, E ). Nevertheless, SA treatment applied by foliar spraying positively modulated the relative expression of CaPOD gene after 28 storage days and, therefore, stimulated the activity of the POD enzyme ( Figures 5F , 6H ). Induction POD activity, which is an important oxyradical detoxification enzyme in plant tissues, has been demonstrated in the present study and may generally facilitate conditions that can delay senescence in green peppers fruits (Zhou et al., 2011). Furthermore, the only relative expression of those antioxidant enzyme activities-related genes that was upregulated after 28 days of storage was for the POD enzyme ( Figure 6H ). These results are in accordance with those reported by Rao et al. (2011) in which 1- and 2-mM SA postharvest treatments extended the shelf-life of sweet pepper fruit (Capsicum annum L., cv. Indra). Senescence is associated with the defensive system, including antioxidant enzymes, such as POD. In the present study, POD was highly correlated (positively, Pearson) with firmness, color, TA, chlorophylls, phenolics and AA content, L-TAA, APX and the relative expression of both CaAPX and CaCAT genes ( Figure 8B ). An efficient antioxidant system can postpone the senescence process even though antioxidative activity in fruits decreases with ageing (Zheng et al., 2007). Antioxidants can delay, retard or prevent oxidation processes by reacting with free radicals, chelating metals and acting as oxygen scavengers, a triplet as well as singlet form and transferring hydrogen atoms to the free radical structure. Similar results were recently reported where the incorporation of SA foliar spraying and caraway oil coating resulted in the highest antioxidant enzyme activity and the lowest chilling injury in treated pepper fruits stored under cold conditions (Hanaei et al., 2022). The results of the present study reinforce the knowledge gap about the effect of SA applied by foliar spraying and irrigation on the complex interaction of metabolites and genes in regulating the bell pepper fruit ripening and senescence processes.

Finally, whilst not the primary focus of this study, it has been demonstrated that the foliar and irrigation application of SA on bell pepper plants has the capacity to enhance fruit quality without exerting a detrimental effect on productivity. The results suggest that both foliar and irrigation SA treatments improve the accumulated crop yield, expressed in terms of kg per plant, throughout the crop cycle of ‘Lamuyo’ green pepper fruit, ‘Herminio’ cv., as it can be observed in Supplementary Figure 1 . However, no significant differences were appreciated between the two application methodologies (p ≥ 0.05). The role of SA in improving fruit yield may have been due to the translocation of more photoassimilates to fruits, thereby increasing fruit weight and reducing negative environmental impacts. These findings confirm those observed in a previous study where an increase of 0.5 kg yield was recorded in the last harvest date after the foliar application of SA (Dobón-Suárez et al., 2021a). Other studies also observed a higher productivity with the preharvest application of exogenous SA in different types of pepper plants (Sobczak et al., 2023; Preet et al., 2023; Ghahremani et al., 2023; Ibrahim et al., 2019; Elwan and El-Hamahmy, 2009).

In summary, the results of the present study demonstrated that both SA preharvest methods tested (foliar and irrigation) were effective in enhancing crop yield, fruit quality at harvest, and delaying ripening and senescence of green pepper fruit throughout the antioxidant metabolism. The effect of both SA applications was found to be similar in most of the parameters analyzed, although it should be highlighted that they showed differences as follows: Firstly, foliar spraying of SA led to a positive modulation of the relative expression of CaPOD and CaPAL genes at harvest, although this stimulation was only upregulated for the CaPOD gene after 28 days of storage. Secondly, irrigation with SA led to an enhancement of H-TAA at harvest and TSS content after postharvest storage. Thirdly, the irrigation method of SA showed a significant increase in CAT activity and delayed weight loss and ripening index. However, regarding their impact on the field and on the agri-food industry, it should be emphasized that the irrigation method was the most beneficial due to the ease and cost-effectiveness with which it could be applied in comparison to foliar spraying.

In our study, we found that salicylic acid (SA) preharvest treatment, both by foliar spraying and irrigation, improved the fruit quality of green peppers, resulting in a slowing of the ripening and senescence processes during postharvest storage, while stimulating the antioxidant system, accompanied by an increase in the content of secondary metabolites and activity of antioxidant enzymes, mediated by the upregulation of the relative response of genes responsible for their biosynthesis. The mechanism of action proposed in the present study is as follows: 1) SA induces the synthesis of secondary metabolites and enhances the antioxidant systems by stimulating the phenylpropanoid biosynthesis pathway, and 2) SA activates the antioxidant enzymes by acting on the modulation of the relative expression of antioxidant system-based genes and regulating the balance between the antioxidant system and ROS metabolism, contributing to the antioxidant metabolism insights. The results of the present study fill the knowledge gap on the effect of SA applied by spraying and irrigation on the complex interaction of metabolites and genes in regulating the ripening and senescence processes of pepper fruit. Finally, both foliar spray and irrigation SA applications showed a great effect on fruit quality and crop yield. However, in terms of beneficial effects on the field and for the agri-food industry it should be highlighted the irrigation method due to the easier way and the reduced cost of application compared to the foliar application.