Biochemical dynamics during postharvest: Highlighting the interplay of stress during storage and maturation of fresh produce

Fresh products

Plants have been a primary source of sustenance for humans since ancient times. Over the years, human development has advanced the processing, preservation, and consumption of these products. Currently, certified organizations, such as the World Health Organization (WHO), widely recommend consuming fresh products (daily intake of 400–600 g of fruit) [7].

Fruits and vegetables are rich in bioactive compounds, including phenols, carotenes, flavonoids, and isothiocyanates among others. These compounds contribute to their nutritional value and provide potential health benefits. It is important to be aware that these claims need further research before definitive conclusions can be drawn [8]. The beneficial effects of these constituents have been subject to comprehensive scientific testing, affirming their antioxidant, anticarcinogenic, and antidiabetic qualities [9,10]. Several researchers are currently investigating the importance of regularly consuming fresh produce within the context of a diet specific to certain diseases. For instance, men who consumed vegetables, fruits, and berries more than 27 times a month showed a 10% lower risk of all-cause mortality and a 20% lower risk of strokes compared to those with lower consumption [11]. Specific bioactive components present in fruits and vegetables prompt their consumption as a solution to health problems. For example, the abundance of anthocyanins in blueberries has demonstrated biomedical benefits such as reducing the risk of cardiovascular diseases, age-induced oxidative stress, and inflammatory responses [12]. Likewise, lycopene found in tomatoes has demonstrated a reduction in the risk of cardiovascular diseases [13]. Furthermore, research has shown that sulforaphane, which is found in Cruciferae vegetables such as broccoli, kale, cabbages, and cauliflower, can help reduce the risk of colon and prostate cancer [14].

Failure to follow proper postharvest practices will result in numerous reactions, such as an increase in respiration rate and ethylene production, acceleration of enzymatic activity (inducing softening by degradation of cell wall components), chlorophyll degradation, and wounding, which may increase bacterial and fungal populations [15,16]. Therefore, proper handling, transportation, and storage during harvest can delay spoilage and prevent postharvest losses.

Quality of fresh products and storage fundamentals

Preserving the quality of fresh produce has posed a challenge to both the horticultural industry and the scientific community. In the early stages of human history, individuals utilized temperature control and stored their products in naturally cooler locations to prolong fresh produce shelf life. However, storage is a complex undertaking that requires careful consideration, as excessively low temperatures can lead to chilling injury [17].

Although temperature control is critical in storage, the use of technologies such as controlled atmospheres (CA), MAP, and coatings is complements used in fresh product industries. Controlled and MAP involve exposing fresh produce to an environment with artificially regulated gas composition, which can be implemented in packaging or storage. This application often involves the reduction of O₂ and CO₂ increment [18]. The use of CA has demonstrated successful results in fresh product preservation through the reduction in respiration rate, microbial growth, chlorophyll breakdown, and enzymatic browning [19]. Likewise, MAP refers to the fresh produce self-creation of an atmosphere utilizing carbon dioxide (CO₂), and ethylene (C₂H₄) produced by biochemical events (e.g., respiration and ethylene production).

Meanwhile, while CA is commonly used for long-term storage, MAP has been proven effective for fresh-cut products, including climacteric ones such as avocados, figs, and tomatoes, as well as, for non-climacteric ones like strawberries and berries [20,21,22].

The combination of these treatments is usually utilized for seeking the prolongation of fresh products shelf-life. For instance, a combination of CA short-term storage (20% CO₂ and 5% O₂) and MAP packaging was applied in haskap, improving the antioxidant activity, and retaining the firmness after 14 days of storage at 2°C/90–92% RH [23]. Additionally, CA (10% CO₂) was effective in delaying the fungus count population in strawberries stored at 5°C for 10 days [24]. Moreover, the use of CO₂ (10 and 20 kPa) and O₂ (10 kPa) CA in five cultivars of highbush blueberry helped in the preservation of fruit quality stored at 1°C for 6 weeks. Although CO₂ has been demonstrated effective results in CA, other shreds of evidence suggest a decrement in quality traits such as firmness and sugar, acid ratio, and the increment in undesirable conditions (e.g., ethanol, acetaldehyde, and ethyl acetate accumulation) [25].

Firmness

Firmness is one of the most relevant factors in human perception of the quality of fresh produce. Accordingly, defining and enhancing this attribute has captivated postharvest researchers. Berries, for example, naturally lose firmness during fruit maturation, such as swift softening in cultivated strawberries that diminishes their shelf-life. [26]. Storage temperature is crucial for firmness preservation. For instance, chili peppers experience less loss of firmness stored at 20°C than when stored at 30°C, likewise, the firmness of blueberries stored at 5°C, maintained greater firmness than those stored at 10°C during 7 days [27,28]. On the other hand, zucchini fruit stored at 4°C shows an increase in the enzymatic activities of pectin methylesterase, polygalacturonase, and cellulase; these enzymes have been related to cell wall deterioration. Likewise, chilling injury and cell wall degradation are related [29].

Postharvest treatments, including the exogenous application of methyl jasmonate (MeJA), salicylic acid (SA), calcium chloride (CaCl₂), and polyamines, have been used to preserve the firmness characteristics of different fruits [30,31,32]. For instance, 10 μM MeJA for 24 h at 20°C treatment has been applied in loquat fruit and then stored at 1°C for 35 days. Firmness preservation in comparison to the control was reached through the reduction of chilling injury [30]. SA is an exogenous treatment related to firmness preservation and the ripening delay through the inhibition of ethylene biosynthesis [33]. Dipping peaches in 1% CaCl₂ solution demonstrated an increment of firmness of the fruit storage (0, 4, and 10°C/95% RH); nevertheless, temperature demonstrated a significant relation to firmness. Higher temperatures lead to a lower firmness in treated peaches [34]. In addition, CaCl₂ treatment has shown the strengthening of the cell wall in litchi fruit (5 g L⁻¹) solution vacuum infiltration (5 min). This property is attributed to an increment of Ca²⁺, water-soluble pectin, and upregulates the activities of pectinesterase and β-galactosidase while decreasing the activities of polygalacturonase and cellulase [35].

Color

Color is a critical factor by which consumers select fresh produce, but it holds more significance than being an organoleptic characteristic. Chemically, pigments like carotenoids can stimulate defense mechanisms in fresh produce and possess powerful antioxidant properties against ¹O₂, primarily due to the conjugated double bond [36]. Likewise, coloration encompasses several physiological and biochemical events mainly related to fresh product ripening. Thus, color is an indicator of the maturity stage of fresh produce. Therefore, the evaluation of color during storage has been reported and is a critical step in determining the effectiveness of postharvest treatment and gaining knowledge of ripening delay. Postharvest treatments are effective in retarding color development, for example, in tomatoes 1-methylcyclopropene (1-MCP) treatment of 250 nL L⁻¹ for 16 h and stored for 18 days at 22°C reached a delay in the color change rate by 4–6 days [37]. Likewise, changes in the color of fresh products involve enzymatic activities mainly by polyphenol oxidase (PPO) and peroxidase generating an enzymatic browning which occurs by exposure to air after cutting, slicing, mechanical damage, and cold storage [38]. Evidence to prevent enzymatic browning with chemical methods (e.g., sodium bisulfite, ascorbic acid, SA, melatonin, and nitric oxide) has been demonstrated [39,40]. For example, sweet cherries treated with melatonin at 100 μM demonstrated a reduction of browning index of 38.5%, in comparison to the control after 45 days of storage at 0°C [41]. Moreover, ascorbic acid at 20 mM treatment has confirmed the alleviation of browning in avocados stored at 4°C/95% RH for 14 days through the decrement of POD and oxidative stress [42].

Flavor and aroma

Flavor and aroma are essential qualities for fresh produce. Although the visual appeal of fruits and vegetables is often what initially attracts consumers, producers must prioritize preserving and improving flavor and aroma during storage [43].

In fruits, sugars including sucrose, glucose, and fructose, and acids such as malic in apples, tartaric in grapes, quinic in cranberries, and citric in citrus and tomatoes, contribute significantly to flavor. It is important to note that additional elements, specifically polyphenols and flavan-3-ols, can also impact taste by introducing bitterness [44]. Conversely, aroma is primarily composed of aldehydes, terpenoids, esters, and alcohols [45]. The macro- and micronutrient composition in fresh produce changes as it ripens and is stored, which results in an altered flavor. During this process, crucial roles in the formation of flavor are attributed to events such as the production of ethylene, intensified respiration, and the development of color. Additionally, the response of fruits and vegetables to abiotic and biotic stress during storage leads to the accumulation of taste and aroma-related metabolites, such as anthocyanins, phenolic acids, and terpenes. It should be noted however that oxidative damage to lipids has the potential to generate off-flavors and aromas in fresh products [46]. For instance, storing mandarins at 2°C can result in the accumulation of terpenes, which may decrease overall flavor [47]. Furthermore, chemical treatments like 1-MCP have been reported to influence the flavor of stored peaches. They can lead to increased sweetness due to a higher accumulation of sucrose, while simultaneously reducing the presence of negative flavor compounds like benzaldehyde and histidine. These outcomes might be attributed to the induction of sucrose synthesis and resynthesis, potentially involving the conversion of reducing sugars to sucrose via sucrose phosphate synthase. Additionally, the transport of cytosolic sucrose to vacuoles is facilitated by the sugar transporter tonoplastic monosaccharide transporter. Genetically, this process is regulated by the expression of genes such as PpSUS4, PpNINVI8, PpSPS3, and PpTMT2 [48].

Weight loss

Typically, most fruits become unsellable after experiencing a decrease in weight of less than 5–10% of their original weight [49]. The main cause of weight loss is water evaporation through transpiration. During this process, water vapor moves from the surface of the fruit to the surrounding air. The gradient between the water vapor pressure and the environment in contact with the fruit is what triggers this event [50]. Due to this, reducing water evaporation in postharvest has been a challenge for postharvest technologists. Reducing weight loss in postharvest treatments has been reported. For instance, in haskap, the use of a CA (20% CO₂ and 5% O₂) reduced the percentage of weight loss after 7 days of storage at 2°C, 1.1% in comparison to the control [23]. Likewise, chemical treatments such as nitric oxide (NO) at 15 μmol L⁻¹, showed reductions in weight loss of 5% after 4 weeks of storage at 0°C and 90% RH in peach fruit [51]. Moreover, the use of H₂S (3 mmol L⁻¹) demonstrated an enhancement in weight loss in passion fruit. The results showed that the control was 6 times higher than the treatment [52].

Fresh products ripening

Fresh produce is distributed in nature and is essential to the human diet. However, consumption limitations arise during the ripening stage due to natural physiological, biochemical, and organoleptic changes [54].

Ripening involves several processes that occur in the latter stages of fresh product growth and the early stages of senescence. The main reactions associated with the ripening process involve a change in membrane permeability, which damages cellular integrity [54]. Management of postharvest systems for understanding ripening in fruits involves the comprehension of physiological, biochemical, and molecular events mainly related to ethylene synthesis [55].

Fresh products are subclassified according to the ripening process; thus, climacteric products are related to dramatic increments in respiration and ethylene production during the ripening process, normally climacterics reach significant ethylene levels which induce biological responses [56]. On the other hand, non-climacterics do not exhibit significant ethylene production, and recent research suggests the central role of abscisic acid (ABA) in non-climacteric ripening [57].

Respiration

The description of cellular respiration is the fundamental energy-conserving process generating adenosine triphosphate (ATP), which can be anaerobic and aerobic. Throughout this process, sugars (mainly sucrose and starch) are reduced into simpler molecules (CO₂ and H₂O) (Figure 1).

Figure 1

Glycolysis equation. Inspired by Copeland and Turner [58]. The glycolysis equation: Glucose + 2 NAD + + 2 ADP + 2 pi 2 → 2 pyruvate + 2 NADH + 2 H + + 2ATP + 2H₂O.

In the process of glycolysis, glucose is subjected to a sequence of enzyme-driven reactions in the cytosol, leading to the formation of pyruvate, ATP, and NADH. Pyruvate enters the tricarboxylic acid (TCA) cycle, where it is converted to acetyl-CoA and further metabolized to generate NADH and FADH₂. In the TCA cycle, acetyl-CoA combines with oxaloacetate to form citrate, which undergoes a series of transformations, including isomerization and decarboxylation, to produce alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, and malate. These reactions result in the generation of ATP, NADH, and FADH₂, providing energy for cellular processes [58,59]. This process generates reducing equivalents which are used by the mitochondrial ETC to perform the synthesis of ATP [60]. During ETC, 0.2–2% of electrons do not follow the process. Instead, a direct leak out of ETC interacts with O₂ to produce ˙ O 2 − and H₂O₂. Thus, plants can modulate the production of ROS through the activation of AOX. Via the activation of AOX, ROS production is minimized through the reduction of ˙ O 2 − , and the dissipation of the electrons flow across the mitochondrial membrane which prevents electron leakage [61].

During the growth of fresh produce, respiration plays a key role in metabolic activities. For example, the development of pigments (e.g., anthocyanins and carotenoids) results in an increase in fruit respiration due to the energy required for biogenesis.

Fresh produce texture can be influenced by high respiration rates which can cause softening due to pectin degradation through metabolic acceleration and induction of cell wall degrading enzymes. In terms of flavor, the changes in total sugar content are affected due to the sugar breakdown to generate CO₂. Additionally, weight loss occurs as energy is consumed through respiration, which utilizes O₂ to produce CO₂ and H₂O. A high respiration rate induces the overproduction of ROS which participates directly in programmed cell death inducing spoilage in fresh produce.

Ethylene as a key phytohormone during ripening

Ethylene is a gaseous plant hormone which is related to fruit ripening, growth, and biological events (e.g., responsiveness to stress and pathogen attack). Moreover, ethylene defines the classification of fresh produce as climacteric. Hence, understanding the ethylene process in fresh products is essential in post-harvesting for determining the optimization of handling, storage, and transportation [62].

Ethylene biosynthesis is catalyzed through two systems, system 1 is an auto-inhibitory phase with basal levels of the phytohormones and reduces C₂H₄ responses. After, a catalytic process, which increases C₂H₄ production, is carried by system 2 [63]. These pathways start with the initial methionine precursor which is converted into S-adenosyl methionine (SAM). Then, a reaction with pyridoxal phosphate to produce a Schiff base forming cyclopropane ring and 5′-methylthioadenosine (MTAN), subsequently a cyclopropane ring is converted to 1-aminocyclopropane-1-carboxylate (ACC) by ACS enzyme which is an essential event for C₂H₄ synthesis. ACC is oxidized by ACC oxidase to form ethylene [64]. The perception of C₂H₄ in the endoplasmic reticulum is carried by transmembrane-receptor proteins [65]. Ethylene perception is classified into two subfamilies, ethylene receptor 1 (ETR1) and ethylene response sensor 1 (ESR1) belong to subfamily-I, whereas subfamily-II contains receptor isoforms ETR2, ERS2, and ethylene insensitive 4 (EIN4). Moreover, a protein kinase constitutive triple response (CTR 1) and ethylene intensive 2 release the transcription factors ethylene intensive 3 (EIN3), EIL1, EIL2, and ERFs, leading to ethylene responses [66].

Various quality parameters, such as fruit firmness, sugar content, acidity, starch, pectin, enzymes, and aroma volatiles, influence the production of ethylene in fresh produce. Furthermore, environmental factors, such as temperature, humidity, and gas composition, significantly impact ethylene biosynthesis. High temperatures raise ethylene production, while high humidity mitigates it [67].

Ethylene triggers the activation of enzymes involved in cell wall degradation, such as polygalacturonase, which induces softening by breaking down cell wall polysaccharides. Ethylene also coordinates ripening, inducing carotenoid biosynthesis, chlorophyll degradation, and starch hydrolysis into simple sugars, resulting in color changes in fresh produce [68]. Moreover, ethylene in conjunction with respiration rate may accelerate metabolic activities through the acceleration of ripening, thus, the higher CO₂ production induces weight loss leading to deterioration in fresh produce quality. Therefore, ethylene content is monitored, and postharvest treatments are focused on its control. For example, the use of edible coatings, such as 1% and 1.5% Tragacanth Gum (TCG), can improve the quality of persimmon fruit by reducing ethylene synthesis. Additionally, abiotic stressors, such as UV-C and blue LED light, can induce ethylene synthesis and alter the quality of fresh produce [69,70].

Abiotic stress

Abiotic stress refers to unfavorable situations (high temperature, salinity, high metals, drought, air pollution, and radiation) that plants must face throughout life [71]. These conditions cause extensive losses in agricultural production. Nevertheless, in fresh products, controlled abiotic stressors are utilized for enhancing and prolonging quality and shelf life; once the stress is applied, fresh produce launches specific defense responses. For example, heat stress induces metabolic changes accumulating heat shock proteins, and the induction of antioxidant systems in tomatoes and broccoli [72,73]. Likewise, the use of chemicals such as exogenous plant growth regulators [e.g., auxins, ABA, gibberellins, jasmonic acid (JA), ethylene, nitric oxide, and SA] can be effective in fresh product stress mitigation [74]. The perception of stress triggers the disruption of functional sensors such as calcium (Ca²⁺), ROS, NO, and phospholipids [75].

The use of abiotic stressors on fresh produce can also trigger genetic responses. In cucumbers, the application of cold temperature (4°C) and MeJA induces CsLOX4 and CsLOX8 gene expression, which are involved in aromatic composition [76]. Abiotic stress perception of fresh products leads to a defense mechanism activation; thus, the enhancement of fresh produce quality can be induced. UV-C and the use of NaHS treatment for H₂S endogenous production will be described below.

Ultraviolet radiation

Radiation is a physical phenomenon which has been utilized in postharvest for enhancing fresh product quality and prolonging shelf-life. In plant physiology, light is a major factor that has a major influence on driving carbon metabolism [77]. Fluorescent lamps produce artificial light and are adjustable for set treatments under different light levels and photoperiods. Ultraviolet light wavelength corresponds to 200–400 nm. In this wavelength range, UV is divided into three subgroups: UV-A (320–400 nm), UV-B (280–320 nm), and UV-C (200–280 nm). These wavelengths have different effects in plants; UV-B and UV-C have been suggested as non-enzymatic and enzymatic antioxidant system promoters (Table 1) [78]. Likewise, light exposure represents a critical factor for fresh products and their perception of stress. Thus, changes in light intensity can lead to acclamatory responses and cellular damage triggering ROS production [88,89].

Chemical postharvest treatments enhance quality and prolong shelf-life

New techniques and the use of chemical solutions to induce defense mechanisms can improve the quality and extend the shelf-life of fresh produce. In addition to physical treatments, the use of 1-MCP demonstrated the inhibition of ethylene delaying ripening in climacteric fruit. Similarly, the use of NO and hydrogen sulfide (H₂S) in fresh produce inhibits postharvest diseases and chilling injury, and the use of growth promoters, such as SA, MeJA, and ABA, as exogenous chemical agents are associated with the accumulation of antioxidants [90].

Although the early stages of chemical treatments were focused on decontamination, current research suggests their application in defense mechanisms (enzymatic and non-enzymatic antioxidants, and genetical induction). For instance, the use of 2 mM SA solution in peaches incremented the inhibition percentage of DPPH radical by 10% after 42 days of storage at 0°C; likewise, phenolic content increment 29 mg in comparison with the control. Moreover, firmness displayed higher values and weight loss percentage was reduced [91]. Chemical treatments have been utilized as inducers of enzymatic and non-enzymatic antioxidant systems. For example, SOD, CAT, APX, and phenolic components have been stimulated by MeJA (50 μmol L⁻¹) in blueberries stored at 22°C for 7 days [92]. Likewise, an emergent chemical treatment utilized for enhancing fresh products is H₂S. In eggplants, the use of 0.1–0.3 mM obtained higher firmness levels, anthocyanins, TSS, and vitamin C accompanied by lower weight loss and chilling injury indices after 21 days of storage at 7°C [93].

H₂S

H₂S is an emerging signaling molecule which induces defense systems against adverse environmental situations such as heavy metals, salinity, postharvest senescence, and biotic stress resistance [94]. H₂S is an endogenous molecule produced in plants; however, the use of sodium hydrosulfite (NaHS) as a donor is used to apply exogenous treatments in the postharvest process [95]. The endogenous production of H₂S in plants involves the participation of sulfite reductase, which catalyzes the reduction of sulfite to sulfide; nonetheless, this process involves two cysteine-dependent reactions and residuals (e.g., β-cynoalanine, synthase + hydrogen cyanide), which produce H₂S through the detoxification of cyanide at the expense of cysteine [96].

In plants, H₂S functions have been described in several processes (e.g., alteration of genes and enzymatic activities, regulation of secondary metabolites) [97]. Recent studies suggest the importance of H₂S during fresh product storage due to the enhancement of the antioxidant system. Meanwhile, the enzymatic antioxidant system enhancement has been reported in broccoli, eggplant, and kiwifruit [98]. The induction of non-enzymatic antioxidant system has been reported through the induction of the glutathione–ascorbate cycle and phenolic content increment [99]. Likewise, the use of NaHS (1.5 mM) treatments resulted in the increment of H₂S in strawberries from 0.71 to 1.81 mmol kg⁻¹.

On the other hand, heat treatments decreased the levels to 0.85 mmol kg⁻¹. Moreover, the increment of H₂S levels stimulated AOX accumulation through the induction of FaAOX3 expression level [100]. Other authors’ hypotheses suggest the induction of AOX through the inhibition of cytochrome oxidase; in the presence of an aerobic system, cytochrome c, and ascorbate, cytochrome oxidase is rapidly inhibited by sulfide. Thus, when cytochrome oxidase is inhibited, the detection of mitochondrial dysfunction releases the AOX pathway [101].

Oxidative stress

Fresh fruits and vegetables are the main sources of antioxidants (e.g., polyphenols, flavonoids, and vitamins) for the human being, nevertheless, a decrease in antioxidants shows up at the harvest and storage stages. Likewise, the generation of ROS throughout these conditions depletes antioxidants through reduction–oxidation reactions [102]. Cellular energy metabolism is based on ATP. During this process, ¹O₂ accepts electrons and H⁺ then is reduced to H₂O [103]. ROS are highly reactive molecules with organic components, and the main ROS transformation involves oxide reduction processes from ¹O₂ until OH˙ (Figure 2).

Figure 2

ROS from singlet oxygen to hydroxyl radical. Inspired by Kamata and Hirata [103]. The mechanism of ROS generation in mitochondria, chloroplasts, and peroxisomes is explained. ROS generation begins with O₂ and ends with the formation of OH in the presence of Fe²⁺ via the Fenton reaction. During this process, both enzymatic (e.g., SOD) and non-enzymatic (e.g., glutathione, ascorbate, and carotenoids) antioxidant defenses are activated to defend the cell. Lipid oxidation, protein oxidation, and DNA damage can result from overproduction of ROS.

Singlet oxygen

Oxygen is a uniquely structured molecule that undergoes serial reactions because of its electron configuration. The continuous spinning of oxygen electrons alters the nature of the molecule and results in transformations to ROS, including ¹O₂ [104]. The ¹O₂ production occurs mainly in the chloroplast, induced by the triplet excitation state of chlorophyll in photosynthesis system two (PSII) at an excitation energy of 94 kJ mol⁻¹ [105]. Thus, ¹O₂ production is mainly related to response under excess light stress when a ³O₂ molecule becomes excited by photosensitizers, two unpaired electrons become paired, and the ground state of ³O₂ turns into ¹O₂ [106].

The complete mechanism of singlet formation was defined by Apel and Hirt [107] and is illustrated in Figure 3. Singlet oxygen acts as a signaling molecule and allows interaction with membrane carotenoids and thylakoid components. Therefore, ¹O₂ is essential for chloroplast nuclear signaling pathways involved in physiological processes such as growth and activation of defense mechanisms.

Figure 3

Reaction center of PSII in singlet oxygen production. Inspired by Apel and Hirt [107]. ¹O₂ is generated using light as input energy, the light absorption by chlorophyll P680 and precursors leads to its generation, likewise, the antenna participates in the formation of a triplet state of chlorophyll (Chl³) through the reaction with triple oxygen state (³O₂) forms a reduction in Chl³ to Chl and a molecule of ¹O_2.

On the other hand, excessive levels of ¹O₂ induce the oxidation of lipids, proteins, and nucleic acids, resulting in cell damage [108]. The double bonds are the primary targets in proteins, specifically amino acids with aromatic ring structures, polyunsaturated fatty acids in lipids, and guanine bases and thiol groups in DNA [109].

Superoxide anion

Overall, ˙ O 2 − is a reduced form of molecular oxygen which is considered a charged anion with a strong oxidant potential. Biologically, this molecule is generated in the mitochondria as a by-product of cell respiration [110]. Superoxide anion is produced by the electron reduction of oxygen and can react with reducing substances or macromolecules in the cell. In addition, ˙ O 2 − can react with SOD to produce H₂O₂ and react with NO to produce peroxynitrite. Superoxide anion is formed by NADPH oxidase and is generated continuously in the photosynthesis system one moreover is produced in the membrane during photosynthesis, and its production triggers the generation of more active ROS such as OH˙. This conversion is described in Figure 4 and involves a system called the Haber and Weiss reaction [111,112].

Figure 4

Haber and Weiss reaction. Inspired by Koppenol [113]. The reaction associates iron with the production of ROS where ˙ O 2 − is a key player participating in two main activities, ferric ion reduces to ferrous ion via the reaction with ˙ O 2 − to turn into OH˙ radicals.

Hydrogen peroxide (H₂O₂)

Hydrogen peroxide is the two-electron reduction product of O₂ and is vastly generated in biological processes acting as a signaling molecule which responds to various stimuli in plants through serial activities, for instance, calcium mobilization, protein phosphorylation, gene expression, and downstream signaling [114,115]. Hydrogen peroxide can diffuse across membranes acting as a signal for fresh product stress perception [116]. However, high concentrations of H₂O₂ have repercussions in plant cells due to its toxicity and induction to ROS generation leading to cell death [117]. In cells, H₂O₂ is produced in peroxisomes, being the photorespiratory glycolate oxidase (GOX) reaction the main event for its production (2- to 50-fold higher than chloroplasts and mitochondria) [118]. The photorespiratory H₂O₂ system has been suggested by Foyer et al. [119] who described the contribution of the H₂O₂ pool to signaling pathways that mediate plant growth and stress responses. Hydrogen peroxide participation in photorespiratory metabolism is described in Figure 5.

Figure 5

Peroxisomal photorespiratory pathway. Model defined by Foyer et al. [119]. In the process of photorespiration, the peroxisomal pathway and its relationship with H₂O₂ as a signaling molecule play a crucial role. During the initial stages of photorespiration, ROS such as H₂O₂ engages in the formation of glycolate oxidase (GO), there is a correlation between the expression of serine: glyoxylate aminotransferase (SGAT) against pathogens and an increase in H₂O₂ yield through the enhancement of GO. Furthermore, glycolate and glyoxylate cycling also contribute to the formation of NADP⁺ and NADPH in conjunction with H₂O₂.

In fresh products, H₂O₂ synthesis is related to the ripening process. In tomatoes, the high light stress (700 μmol m⁻² s⁻¹) promoted the accumulation of H₂O₂ and the induction of ripening due to ethylene interaction [120]. In cucumber stored at 5°C for 12 days, the exploration of H₂O₂ excess has been associated with the induction of chilling injuries and the use of postharvest treatments with 1 μm MeJa and 1 mM NO with diphenyl iodonium 5 mM (DPI) reached promising results reducing about 50% of the chilling injury in comparison with the control [121].

Hydrogen peroxide functions as a signaling molecule by interacting with phytohormones, including SA, JA, ethylene (ET), auxins (AUX), and ABA, to synergistically mediate physiological processes, such as auxin conjugation [122].

Non-enzymatic antioxidants

Antioxidant defense systems in fresh products do not only involve enzymes, which have been described before. Fresh products avoid cell death generated by oxidative stress excess using non-enzymatic antioxidants in berries phytochemicals (e.g., phenolic compounds, and ascorbic acid), and glutathione [123]. Waśkiewicz et al. [124] described the role of non-enzymatic antioxidants in plants and the mechanism against ROS. Further sections provide detailed explanations of these mechanisms.

Phenolic compounds

Phenolic compounds are the widest metabolites found in fruits. Phenolic acids, flavonoids, and anthocyanins are studied due to their influence on fruit quality parameters, principally flavor, color, and aroma [125]. A phenolic in terms of chemistry involves components with at least one aromatic ring (C₆) bearing one or more hydroxyl groups, and their solubility is according to their structure complexity [126]. These secondary metabolites are biosynthesized using essential carbon sources and frameworks, principally the glycolytic pathways of phosphoenolpyruvate, and pentose phosphate in the process of the decomposition of sugar. In addition, the shikimate pathway involves the production of aromatic amino acids such as l-phenylalanine (l-Phe), l-tyrosine, (l-Tyr), and l-tryptophan (l-Trp) which are involved with secondary metabolite generation mainly l-Phe and l-Tyr are present in phenolic pathways such as flavonoids, coumarins, chalcones, and lignans generation [127]. Phenol component’s behavior throughout storage has been deeply studied by postharvest researchers due to their capacity to mitigate ROS. The essential function against oxidative stress is attributed to the reduction of H₂O₂ to form singlet O₂ through the constitution of a phenylpropanoid group with aromatic rings of one or more OH⁻ groups generating a chemical bonding with H₂O₂ [128]. External chemical treatments and stressors for the stimulation of phenolic compounds in postharvest have been studied. Postharvest accumulation of phenolics by applying light stressors has been reported. For instance, tomatoes exposed to UV-C (4 and 8 kJ m⁻²) and then stored at 14°C, 95% RH for 35 days increased gallic, p-coumaric, syringic, and chlorogenic acids. Consequently, the antioxidant capacity was enhanced [129,130].

AsA–GSH cycle

Glutathione is defined as a tripeptide synthesized from cysteine, glutamate, and glycine. In fresh product defense, GSH is a major low-molecular-weight thiol tripeptide in plant tissues [131]. GSH acts against ROS reducing their generation by a disulfuric bond between two glutathione molecules to form its oxidized form [132]. Early research suggested GSH as the major cellular thiol with active cellular redox participation [133]. Further research suggested the key role of GSH in the AsA–GSH cycle as a center reaction in the antioxidant system of defense in plants; subsequently, this concept was transferred to postharvest [134]. This concept will be defined further. Overall, the GSH concentration is found in cells cytosol, mitochondria, and endoplasmic reticulum, and its biosynthesis involves a downstream of biochemical reactions based on two enzymatic steps using ATP and the constituent amino acids [135]. Due to this feature, GSH has been applied as an exogenous treatment triggering an enhancement of postharvest traits in fresh products. For example, a study on bell peppers showed that quality improved when treated with GSH (0.05% w/v) through a sprayed application. The peppers were then stored at 4°C with 80–85% relative humidity for 25 days. The treatment showed a reduction in chilling injury postponing a sharp increment for 10 days and reaching a decrement of 40% after 25 days of storage. These results suggested an enhancement in the bell peppers’ antioxidant defense system [136].

Ascorbic acid (ASC) has wide functions in biological processes (e.g., stress resistance, cell expansion division, and light protection). ASC is synthesized in the d-mannose/l-galactose pathway and its accumulation involves a balanced result of biosynthesis, oxidation, and recycling [137]. Moreover, the conversion of ascorbate to mono-dehydroascorbate by APX has a huge contribution to maintaining cellular redox homeostasis under stress conditions [138]. Under stress, ASC plays a key role in fruits and its effects have been demonstrated (e.g., cherry tomatoes under cold stress, peaches under heat air exposure and hypobaric condition, likewise in the application of UV-B radiation in lettuce) [139,140]. Ascorbate (AsA) which is the reduced form of ACS, is an essential factor in the activation of the AsA–GSH system and its participation in plant cells involves the following steps: APX uses two ascorbate molecules to convert H₂O₂ to H₂O and produce MDHA. MDHA can be directly reduced to ascorbate with the help of MDHA reductase and DAHR, using GSH as a reducing agent. GSH is also regenerated by NADPH-dependent GR from oxidized GSSG. This process is critical for scavenging H₂O₂ and maintaining AsA and GSH in different cellular compartments [141]. Although AsA and GSH participate in the cycle, the influence of environmental factors and molecular repercussions are different. While AsA synthesis and accumulation are related to light reception, GSH responds with higher irradiances which are not responsive to ascorbate contents (Figure 6) [142].

Figure 6

AsA–GSH. Inspired by Foyer and Noctor [142]. Schematic illustration of AsA–GSH cycle, enzyme participation GR glutathione reductase, DHAR dehydroascorbate reductase, MDHAR, monodehydroascorbate, APX ascorbate peroxidase. The final product of the cycle is the reduction of H₂O₂ to H₂O through APX.

SOD

The role of SOD is to function as a defense mechanism by catalyzing the disproportionation of superoxide to molecular oxygen and peroxide. Since SODs play a crucial role in safeguarding cells against toxic molecules produced during aerobic respiration, they have a prominent protective response [143]. SODs were recognized as a group of metalloproteins that act in all oxygen-metabolizing cells and all sub-cellular compartments, their localization is accorded to the metal co-factor [144]. Moreover, SOD is responsible for the conversion of ˙ O 2 − into less harmful products for plants.

In fresh products, SOD activity demonstrated an increase after an atmospheric cold plasma for 60 s in blueberries, likewise, during apple senescence SOD increased in contrast to the healthy apples [145]. The use of exogenous treatments seeking the reduction of oxidative stress through increased SOD activity has been used by postharvest researchers to extend the fresh products’ shelf life. Ding et al. [146] utilized exogenous oxalic acid and SA to prevent chilling temperature stress. In comparison with the control group, SOD activity was higher in treated mangos. The oxalic acid represented the highest activity, these results enhanced fruit tolerance to low-temperature stress. Likewise, MeJA (0.1 mmol L⁻¹) treatment on kiwifruit achieved an enhancement of SOD activity through the AcSOD genetic expression. Generally, SOD is associated with enhanced resistance leading to improved defenses and postharvest features in kiwifruit [147]. Melatonin treatment has been reported to enhance SOD activity and contributed to the decay browning and senescence in litchi fruit, 0.4 mM of melatonin treatment was applied, and then, litchis were stored at 25°C for 8 days [148]. Gibberellic acid has been utilized as an exogenous source for enhancing fresh quality. In Toon Sprout, this treatment enhances the quality through SOD activity increments during postharvest in short-term cold storage for 5 days [149]. SOD activity enhancement in fruits by chemical treatments is described in Table 1.

CAT

CAT is present in all aerobic organisms and is a key player in catalyzing H₂O₂ into water and oxygen. Due to this feature, it is a key player in the regulation of stress in plants [150]. Also, CAT plays a role in reducing the overall level of ˙ O 2 − and maintaining cellular homeostasis under normal growth conditions; thus, CAT reduction increases the sensitivity of plants to oxidative stress [151]. In fresh products such as strawberries, blueberries, litchi, apples, and oranges, CAT activity increase is a signal of stress response enhancement [152,153]. Given this significance, postharvest has led to insights into the regulation of treatments to increase CAT activity. A chitosan treatment in sweet cherry fruit was reported by Pasquariello et al. [154]. The cherries were immersed in a 0.5% chitosan solution for 60 s and stored at 2°C for 14 days. The cherries showed an increase in CAT activity. Additional treatments are outlined in Table 2.

APX

The role of APX is crucial for scavenging ROS, particularly under abiotic stress conditions.

APX is involved in the AsA–GSH and utilizes ascorbate for donating electrons to reduce H₂O₂ to water [155]. APX is the first enzyme involved in the AsA–GSH. APX thus prevents the accumulation of H₂O₂ at toxic levels for cells [156]. APX activity has been reported against stressors such as drought, temperatures, water deficit, salinity, and UV radiation [157]. Due to this, the increment of APX during postharvest is an indicator of the oxide reduction state of cells during storage. In mume fruit (Prunus mume), Imahori et al. [158] reported an increment of APX activity during storages at 1 and 6°C for 15 days. Fruit stored at 6°C showed lower activity of APX compared to fruit stored at 1°C. Although chilling injuries occur frequently according to the decrement of temperature, the combined action of antioxidant enzymes resulted in minor chilling injuries stored at 1°C. Other examples previously described exogenous postharvest treatments for improving antioxidant enzymes. APX enhancement has been reported utilizing chitosan coating in loquat fruit (1% w/v) at 7°C for 21 days. APX enhancement was reached. APX activity enhancement in fruits by chemical treatments is described in Table 2.

GR

GR is a flavoprotein that belongs to the family of NADPH-dependent oxidoreductase and plays a huge role in focusing on plants’ defense against oxidative stress through the scavenging of ROS [159]. GR in collaboration with APX has a key role in AsA–GSH cycle. GR acts in the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH [160]. Harshavardhan et al. [161] described the catalytic activity of GR. In the first step, NADPH acts as a reducing agent of the flavin moiety, which is then oxidized. This leads to a redox activation of the disulfide bridge, resulting in a thiolate anion and a cysteine. In the second step, GSSG is reduced through a thiol-disulfide interchange reaction. The reduction of enzymes is re-oxidized by GSSG. GR is predominantly found in the chloroplast. Nevertheless, isoforms have been found in the cytosol, mitochondria, and peroxisomes [162]. In postharvest, ozone (O₃) has been involved with the activation of the AsA–GSH cycle. Doses of 6.432, 10.720, and 15.008 mg m⁻³, 4°C were evaluated for 0, 14, and 42 days in cantaloupes. The increment of GR activity in treated samples has been related to the enhancement of AsA–CSG as a defense system [163]. The addition of ascorbic acid and oxalic acid (40 and 2 nmol L⁻¹) to a CA (5% CO₂ and 1% O₂) resulted in an increase in GR during 28 days of storage in litchi fruit. This also led to a decrease in H₂O₂, which prevented cell leakage and malondialdehyde (MDA) accumulation [164]. Likewise, NO treatment has delayed winter jujube ripening, 20 μl L⁻¹ for 3 h and stored at 0°C for 75 days. GR activity reached a sharpening increment over 45 days (11–15 U kg⁻¹) as well as, other antioxidant enzymes such as SOD, CAT, and APX demonstrated an enhancement in their activities. In contrast, ROS (H₂O₂, ˙ O 2 − ) decreased significantly [165]. GR activity enhancement in fruits by chemical treatments is described in Table 2.

The role of AOX in fresh products

In plants, AOX is oriented for the oxidization of ubiquinol to the four-electron reduction of O₂ to H₂O; this process is conducted by AOX bypassing proton-pumping through the complexes III and IV in mitochondrial cellular respiration. Meanwhile complexes I, III, and IV of the respiratory chain transport electrons through translocation of protons across mitochondrial membranes inducing ATP synthesis, AOX is not coupled with ATP synthesis and energy accumulation for electron transporting (Figure 7) [101]. Furthermore, the suggestion of AOX induction by altering the cytochrome pathway has been a trend in understanding the stimulation of AOX and plant signaling coordination for altering the respiration pathway. Likewise, AOX activity has been related to maintaining cellular homeostasis through detoxification of ROS overproduction.

Figure 7

AOX activity in the ETC. Model proposed by Vanlerberghe [101]. Membrane respiration change and AOX participation catalyzing cyanide-resistant reduction of oxygen to water without the translocation of protons across the mitochondria membrane, this process acts as a non-energy conserving respiration EFC, complexes are I: NADH–ubiquinone oxidoreductase, II: succinate–ubiquinone oxidoreductase, III: ubiquinol–cytochrome c oxidoreductase, IV: cytochrome c oxidase, and V: ATP synthase.

The alteration in the ETC in the mitochondrial membrane induces the regulation of AOX relating to enhanced cyanide-resistant respiration; under adverse environmental conditions total state of respiration is altered through the inhibition of electron transfer, thus, excess ROS are produced. The activation of AOX allows the consumption of excess molecular oxygen and reduces the excessive ROS accumulation. For instance, the enhancement of AOX induction has reached the decrement of ˙ O 2 − and H₂O₂ in papaya stored at 1°C for 60 days through the enhancement of cyanide-resistant pathway. In addition, the cytochrome pathway was suppressed [177].

It has been proposed by a number of researchers that AOX is activated when mitochondria are under antagonism stimulus, such as biotic/abiotic stress (e.g., salinity, drought, high light, and chilling). Likewise, the high production of ROS generated by stressors can lead to the AOX activation for alleviating oxidative damage in mitochondria [178]. Nevertheless, the general role of AOX is to provide alternatives in energy production.

AOX is encoded by a small group of genes divided into two subfamilies, Aox1 and Aox2, meanwhile, Aox1 induction is related to stress responses (e.g., Aox1a, Aox1b, and Aox1b), the Aox2 genes are mainly related to constitutive or developmentally regulated pathway [179]. For instance, in sweet potatoes stored at 4°C 90% RH, the upregulation of IbAox1 and IbAox2 mediates the redox homeostasis and proline accumulation being induced by progesterone treatment (100 mmol L⁻¹). The first 7 days of storage demonstrate the maximum point of IbAOX1 expression and AOX (0.30 U g⁻¹) activity. On the other hand, IbAOX2 had a sharp increase after 14 days of storage [180].

Recapitulation

Proper postharvest handling of fresh produce is crucial to prevent losses. Developing effective strategies for harvest, transportation, and storage is particularly challenging for crops that are not well-studied. While temperature and relative humidity are the key factors that delay fresh product senescence, advances in technology have introduced novel techniques that involve controlled and MAP, edible coatings and films, the use of postharvest treatments such as chemicals (e.g., NO, SA, MeJA, H₂S), and physical treatments such as heat, high light, UV-A, UV-B, and UV-C, which focus primarily on mitigating oxidative stress in fruits and vegetables. These treatments have been shown to improve quality and prolong shelf-life by activating antioxidant enzymes (i.e., SOD, APX, GR, and CAT) and a non-enzymatic system (i.e., phenolics, flavonoids, anthocyanins, glutathione, and ascorbate). This opens new possibilities for emerging crops that often experience inadequate postharvest handling.

An emerging area of postharvest research is the use of controlled stress doses on fruit. Fresh produce undergoes biochemical changes when subjected to stressors. For example, treatments with 1-MCP and NO inhibit ethylene biosynthesis, which delays ripening. In the same vein, NaHS can function as a donor for producing endogenous H₂S, thereby mitigating ROS production in the ETC across the mitochondrial membrane and activating the AOX pathway. Furthermore, UV radiation results in an overproduction of ROS, leading to the activation of AOX in ETC.