Yifan Yan
Gale G. Bozzo- Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada
Senescence of broccoli is accelerated with temperature abuse (i.e., exposure to warmer temperature than that considered optimal to preserve quality) in the postharvest supply chain. We investigated the impact of exogenous ergothioneine on the senescence of broccoli during temperature abuse storage. Broccoli heads were vertically oriented in immersion tanks so that the bottom of the stalk of each inflorescence was partially submerged in 0, 20, 100, or 500 µM ergothioneine prior to storage at 10°C for 10 days. Metabolite analysis revealed that the exogenous ergothioneine was taken up from the submergence solution and accumulated within the broccoli florets, with the greatest absorption in the 500 µM treatment. Floret yellowing due to chlorophyll loss was limited in broccoli treated with 20 µM ergothioneine relative to all other treatments. By the end of the storage period, H2O2 concentrations in the florets of 100 µM and 500 µM ergothioneine-treated broccoli matched those of the untreated broccoli and were up to 63% greater than the broccoli submerged under 20 µM ergothioneine. By the end of the storage period, broccoli treated with 20 µM ergothioneine had greater ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulfide relative to all other treatments. Broccoli submerged in 20 µM ergothioneine had greater activities of glutathione reductase and dehydroascorbate reductase and a smaller increase in ascorbate peroxidase activity relative to the other pre-storage submergence treatments. The findings of this study provide a feasible postharvest handling strategy for the application of a critical 20 µM exogenous ergothioneine concentration that delays broccoli senescence during temperature abuse storage by preserving the status of endogenous antioxidants.
1 Introduction
Broccoli (Brassica oleracea var. italica) is one of the most widely grown green vegetables in the world. Between 2019 and 2023, approximately 50% of the broccoli in the Canadian retail sector, household and service sector was wasted (Statistics Canada, 2024). Postharvest losses of broccoli occur during grading and in the retail sector (Porat et al., 2018). Maximum shelf-life of broccoli is achieved with storage at 0°C, but shelf-life tends to be shorter in commercial operations due to sub-optimal temperature management practices. For example, large temperature ranges (e.g., –1°C to 19°C) can occur in standard vegetable display cases that are used in commercial operations (Nunes et al., 2009).
Broccoli senescence is associated with yellowing and loss of green chlorophyll pigmentation within the florets (Luo et al., 2020; Ahlawat et al., 2022). Yellowing is more apparent with temperature abuse (i.e., warmer temperature than that which is optimal to maintain quality). Full disappearance of chlorophyll occurs in broccoli after 5 days at 25°C, whereas a higher degree of chlorophyll is preserved at 10°C, and more so at 4°C (Gao et al., 2018). The increased expression of senescence associated genes (e.g., SAG12) is more accelerated in broccoli heads stored at 25°C relative to those held at 4°C (Ahlawat and Liu, 2021). Moreover, there is evidence that senescence of broccoli is dependent upon the buildup of reactive oxygen species (ROS) such as H2O2 and the dysfunction of the ascorbate-glutathione (GSH) recycling pathway (Mori et al., 2009; Raseetha et al., 2013; Li et al., 2022). The antioxidants ascorbate and GSH donate electrons to ROS as a mechanism to limit oxidative stress-related cellular damage. Moreover, ascorbate and GSH detoxify ROS via enzymatic steps (e.g., ascorbate peroxidase, APX) in the ascorbate-GSH recycling pathway, which is dependent upon the availability of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (Lum et al., 2016; Foyer and Kunert, 2024). The interactions of ascorbate and GSH with ROS yield their oxidized counterparts dehydroascorbate (DHA) and glutathione disulfide (GSSG) (Chapman et al., 2019). Ascorbate and GSH losses occur in senescing broccoli. For example, Mori et al. (2009) found that broccoli exhausts 70% of its GSH pool within 1 day of storage at 20°C, and most of its ascorbate pool after an additional 3 days. In other vegetables such as arugula, ascorbate concentrations are reduced with cold storage, whereas there is an accumulation of DHA (Sivakumar and Bozzo, 2024). Broccoli florets immersed in a solution of 3.25 mM GSH accumulate less H2O2 and lose smaller proportions of ascorbate and GSH during 10°C storage relative to untreated florets (Jiao et al., 2025).
Ergothioneine (EGT) is a histidine-derived thiol antioxidant that is less prone to autooxidation than GSH (Cheah and Halliwell, 2012). EGT scavenges hydroxyl radicals and chelates metals such as Fe2+ and Cu2+ that would otherwise mediate the conversion of H2O2 to the more reactive hydroxyl radical (Kitsanayanyong and Ohshima, 2022). EGT primarily occurs in culinary mushrooms. Although EGT is not synthesized in plants, it occurs in plants (e.g., asparagus) that form symbiotic associations with mycorrhizal fungi (Carrara et al., 2023), albeit at levels that are up to two orders of magnitude lower than the concentrations in culinary mushrooms (Sivakumar and Bozzo, 2023). EGT limits melanosis in meat, fish and shellfish products including salmon (Pahila et al., 2017; Kitsanayanyong and Ohshima, 2022), crab and shrimp (Encarnacion et al., 2011, 2012). Button mushrooms sprayed with 120 µM EGT brown less and contain more antioxidants following storage at 4°C for 17 days (Qian et al., 2021). Although relatively fewer studies have investigated the postharvest efficacy of exogenous EGT on horticultural crops, it is known that plants are efficient at absorbing exogenous EGT. Kataoka et al. (2025) revealed that Japanese mustard seedlings absorbed EGT from a hydroponic solution containing a small concentration of EGT (i.e., 4.3 µM). Recently, Gu et al. (2025) reported that browning of sliced potato is limited following immersion in an EGT solution. Moreover, it is known that leaf yellowing of arugula is limited when fully immersed in 100 µM EGT prior to storage at 10° C (Sivakumar and Bozzo, 2024). It is important to note that the aforementioned studies involved the application of exogenous EGT on foodstuffs (e.g., leaves) with a high surface area to volume ratio, but there is no information on whether EGT limits oxidative stress-related senescence of bulky commodities (e.g., low surface area to volume ratio), including inflorescence vegetables such as broccoli.
Here we investigated the effect of partial submergence of broccoli in a solution of EGT (0 µM, 20 µM, 100 µM or 500 µM) on the dynamics of senescence of this inflorescence vegetable including floret yellowing when stored at 10°C, and the relationship to shifts in oxidative stress metabolism. Partial submergence has been used for separate hot water and sucrose treatments of postharvest broccoli (Perini et al., 2017; Xu et al., 2016). The partial submergence approach used in this study differs from other studies where vegetables were fully immersed/dipped in a solution of EGT (Sivakumar and Bozzo, 2024; Gu et al., 2025). In addition, the partial submergence limits wetting of the whole broccoli head and any prolonged drying of the whole vegetable that would be required thereafter.
2 Material and methods
2.1 Chemicals and materials
Unless otherwise mentioned all reagents were purchased from Millipore-Sigma (Oakville, ON, Canada). HPLC consumables and solvents were from Agilent Technologies (Mississauga, ON, Canada), and Fisher Scientific (Mississauga, ON, Canada), respectively.
2.2 Partial submergence treatments and postharvest storage of broccoli
For each of three separate experiments, a minimum of 30 kg of broccoli (B. oleracea L. var. italica cv. ‘Imperial’) heads were harvested from commercial operations within a 200 km radius of the University of Guelph (Guelph, ON, Canada). Broccoli heads were harvested on September 11, September 16 and October 7 in 2024. At harvest, broccoli heads were packed on ice and transported immediately to the University of Guelph (Figure 1). Thereafter, the bottom 5 cm portion of the stalk was removed from each broccoli head prior to transferring to a 66-L plastic tub (66.4 cm × 41.3 cm × 34.3 cm, length × width × height, outer dimensions at the top of the container; Sterlite Corporation, Townsend, MA, USA) containing 7.5 L of the freshly prepared EGT solution. A minimum of 15 broccoli heads were transferred to each of four plastic tubs containing 0 µM EGT (control, MilliQ water), 20 µM EGT, 100 µM EGT, and 500 µM EGT, respectively. For the partial submergence treatments, EGT was sourced from Fisher Scientific Canada (Mississauga, ON, Canada). An additional source of EGT was from BOC Sciences (Shirley, NY, USA). The heads were vertically oriented with the bottom 3.5 cm portion of the stalk partially submerged under the EGT solution. The containers were sealed and left at 4°C in the dark. The broccoli heads were withdrawn from the submergence solution after 24 h and the cut end of the stalk was blot dried. The broccoli heads of each treatment were transferred to separate plastic storage bins (60.64 cm × 40.32 cm × 41.59 cm, length × width × height; United Solutions, Leominster, MA, USA) and stored at 10°C and relative humidity of 92–94% under darkness. Three randomly selected broccoli heads were removed from each treatment replicate on each of days 0, 3, 7 and 10 of storage. The fresh weight (FW) of each broccoli head was recorded following the removal from storage. Thereafter, the broccoli heads were dissected to remove the florets from the stalk. For each EGT treatment replicate, the florets of the three separate broccoli heads were pooled. The broccoli materials were flash frozen and pulverized under liquid N2 and held in an ultra-low cryogenic freezer. The frozen florets were used for all biochemical analyses as described under sections 2.3 to 2.7.
Figure 1. Schematic representation of a representative experiment that tested the impact of pre-storage application of exogenous ergothioneine (EGT) and subsequent storage at 10°C on fresh broccoli heads. Created in BioRender. Bozzo (2025) https://BioRender.com/q3grohg.
2.3 Floret chlorophyll analysis
Chlorophyll concentrations were determined as described previously (Lichtenthaler and Buschmann, 2001). Specifically, 120 mg of frozen floret powder was resuspended with 1.5 mL of pre-chilled 80% (v/v) acetone, vortexed for 1 min, and then clarified by centrifugation at 5,000g for 5 min. The pellet was re-extracted up to two more times until it was devoid of green color. A 1 mL aliquot of the supernatants pooled from the successive acetone extracts was transferred to a 1-mL quartz cuvette, and absorbance was monitored at 647 nm and 663 nm with a BioTek Epoch 2 spectrophotometer (Fisher Scientific Canada). For each EGT treatment replicate, chlorophyll a and b within the extract was based on mathematical formulae as described by Lichtenthaler and Buschmann (2001) and expressed on a FW basis.
2.4 High-performance liquid chromatography-tandem mass spectrometry analysis of floret ergothioneine concentrations
EGT was extracted from the florets and analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) as described by Carrara et al. (2023), with a few modifications. For each EGT treatment replicate, 1 g of frozen broccoli powder was combined with 5 mL of pre-chilled 80% (v/v) HPLC-grade methanol, and shaken on a vortex platform for 10 min. The homogenate was clarified by centrifugation at 3,000g for 10 min and then passed through a 0.45 µm polytetrafluorethylene (PTFE) syringe filter (Mandel Scientific Company, Guelph, ON, Canada). A 10 µL aliquot of the filtered extract was injected on to a Zorbax RX-SIL column (2.1 mm × 150 mm, 5 µm) (Agilent Technologies) pre-equilibrated with a mixture of 10% solvent A (15 mM ammonium formate) and 90% solvent B (acetonitrile) connected to an Agilent 1260 Infinity HPLC. EGT was eluted at a flow rate of 0.6 mL min–1 using a gradient as follows: 90% solvent B (0–0.5 min), 90–10% solvent B (0.5–5 min), 10% solvent B (5–5.5 min). Thereafter the column was returned to 10% solvent A and 90% solvent B using a linear gradient over 0.5 min and held at 90% solvent B for an additional 3 min. The column was thermostatted at 30°C. EGT was eluted at a retention time of 4.50 min.
Tandem mass spectrometry (MS/MS) was performed on a Bruker EVOQ® triple quadrupole detector (Bruker Ltd., Milton, ON, Canada). The mass spectrometer was operated in positive electrospray ionization mode at a collision energy of 12 V, spray voltage of 4000 V, cone and probe temperature of 350°C, cone gas flow rate of 20 L min–1, probe gas flow rate of 40 L min–1, and nebulizer gas flow of 60 L min–1. The parent ion (m/z 230.3) and fragment ion (m/z 127.1) of EGT were monitored using multiple reaction monitoring mode and a scan time of 66.7 ms. Data acquisition was conducted with a Bruker Daltonics MS-Workstation (version 8.2.1), using a signal-to-noise ratio of 10 for quantification of the EGT fragment ion peak. For quantification of EGT in broccoli florets, the respective peak areas within the methanolic extracts were compared to a known range (0.03125–100 pmol) of an authentic EGT standard (≥ 98.0% purity).
2.5 Determination of hydrogen peroxide (H2O2) concentrations in stored broccoli
The extraction of H2O2 from the florets of each EGT-treatment replicate was as described by Mátai and Hideg (2017) with a few modifications. Briefly, 250 mg of frozen broccoli powder was combined with 1 mL of pre-chilled 6% (w/v) trichloroacetic acid and vortexed for 3 min. The homogenate was centrifuged at 13,000g and 4°C for 5 min, and recentrifuged as described above if necessary. A 180 µL aliquot of the clarified extract was neutralized to pH 7.0. H2O2 was quantified with the Amplex™ Red hydrogen peroxide/peroxidase assay kit (Fisher Scientific Canada) as per the manufacturer’s instructions with a few modifications. Each 100 µL assay contained final concentrations of 50 mM Na2HPO4: NaH2PO4 (pH 7.4), 25 µM Amplex™ Red reagent, 0.5 milliunits of horseradish peroxidase and the neutralized extract. All microplate assays were initiated by the addition of the neutralized extract (40 µL). Thereafter, the plate was transferred to a BioTek Epoch 2 spectrophotometer, shaken for 5 s and the absorbance was monitored at 570 nm for 4 min. For each broccoli floret extract, the absorbance change at 570 nm was compared to those generated for a known range (0.05–2 nmol) of a H2O2 standard.
2.6 Analysis of ascorbate, glutathione and pyridine dinucleotide profiles in stored broccoli
The floret ascorbate concentration was determined as described previously (Sivakumar and Bozzo, 2024). For each treatment replicate, 200 mg of frozen powder was homogenized with 100 mg of acidified silica sand and 1 mL of 6% (w/v) meta-phosphoric acid using a pre-chilled mortar and pestle. The homogenate was left on ice for 3 min in the absence of light and then centrifuged at 13,000g and 4°C for 10 min. Thereafter, the supernatant was transferred to a pre-chilled microfuge tube, left on ice in darkness for 15 min, and then passed through a 0.45 µm PTFE filter. A 5 µL aliquot of the filtered meta-phosphoric acid extract was injected onto a Restek™ Ultra Aqueous C18 column (150 mm × 4.6 mm, 5 µm, 100 Å) (Fisher Scientific Canada) attached to an Agilent 1100 HPLC coupled to a diode array detector and eluted with 20 mM ortho-phosphoric acid as described by Sivakumar and Bozzo (2024). Total ascorbate (i.e., sum of ascorbate and DHA) of each floret sample was determined following the incubation of the meta-phosphoric acid extract with 0.5 volume of dithiothreitol (DTT) as described by Sivakumar and Bozzo (2024). The ascorbate in each DTT-treated and non-DTT treated extract was detected at an absorbance of 254 nm, and the peak area and retention time were compared to those of a known range of an ascorbic acid standard (0.1–10 nmol). The DHA concentration within each floret sample was calculated by subtracting the reduced ascorbate concentration from the total ascorbate concentration.
GSH and GSSG concentrations within florets were measured spectrophotometrically with a glutathione reductase (GR)-coupled assay that reduces 5,5-dithio-bis-(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid (Queval and Noctor, 2007). The preparation of the neutralized extract and the microplate assay were as described previously (Sivakumar and Bozzo, 2024). To determine total glutathione, each 200 µL assay contained 0.1 M NaH2PO4 (pH 7.5), 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM NADPH, 0.6 mM 5,5-dithio-bis-(2-nitrobenzoic acid), 0.2 units GR, and 20 µL of the neutralized extract. The rate of 5-thio-2-nitrobenzoic acid formation was monitored at an absorbance of 412 nm for 3 min and compared to rates generated for a known range (50–1000 pmol) of GSH standard. For GSSG quantification, the GR-based assay was performed on the neutralized extract following incubation with 2-vinylpyridine as described by Sivakumar and Bozzo (2024). For extracts incubated with 2-vinylpyridine, 5-thio-2-nitrobenzoic acid formation rates were compared to rates generated for a known range (20–500 pmol) of GSSG standard. The GSH within each extract was calculated as the difference between total glutathione and GSSG.
The floret concentrations of non-phosphorylated and phosphorylated pyridine dinucleotides were determined with a recycling assay that detected the phenazine methosulfate-based reduction of dichlorophenolindophenol as described by Queval and Noctor (2007) with a few modifications. Briefly, for nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) determinations, 100 mg of frozen floret tissue was combined with 1 mL of 0.2 N HCl, vortexed for 5 min and centrifuged at 13,000g and 4°C for 10 min. A 100 µL aliquot of the supernatant was incubated at 90°C for 1 min and then chilled on ice. Thereafter, the pH was adjusted to between 5.0 and 6.0 with 100 µL of 0.2 N NaOH and 40 µL of 0.2 M NaH2PO4 (pH 5.6). For reduced nicotinamide adenine dinucleotide (NADH) and NADPH determinations a duplicate floret subsample was extracted with 0.2 N NaOH as described above and incubated at 90°C for 10 min. The extracts were then chilled on ice, and the pH was adjusted to between 7.0 and 8.0 with 75 µL of 0.2 N HCl and 30 µL of 0.2 M NaH2PO4 (pH 5.6). For NAD+ and NADH assays, 20 µL (for NAD+ assay) or 30 µL (for NADH assay) of the neutralized sample extract was combined with 90 µL of assay mix consisting of 50 µL of 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) with 2 mM EDTA (pH 7.5), 20 µL of 1.2 mM 2,6-dichlorophenolindophenol, 10 µL of 20 mM phenazine methosulfate, and 10 µL of 2,500 units mL–1 aldolase dehydrogenase. The reaction was initiated by adding 90 µL of diluted ethanol consisting of 50 µL of 0.1 M HEPES containing 2 mM EDTA (pH 7.5), 15 µL of absolute ethanol, and 25 µL of MilliQ water. The reaction was monitored at an absorbance of 600 nm for 2 min with a BioTek Epoch 2 spectrophotometer. For NADP+ and NADPH assays, 20 µL (for NADP+ assay) or 30 µL (for NADPH assay) of the neutralized sample extract was combined with 90 µL of assay mix consisting of 50 µL of 0.1 M HEPES containing 2 mM EDTA (pH 7.5), 20 µL of 1.2 mM 2,6-dichlorophenolindophenol, 10 µL of 20 mM phenazine methosulfate, and 10 µL of 10 mM glucose-6-phosphate. The reaction was initiated by the addition of 90 µL of a glucose-6-phosphate dehydrogenase (G6PDH) working solution. The G6PDH working solution was prepared by mixing 50 µL of 0.1 M HEPES containing 2 mM EDTA (pH 7.5) with 10 µL of 200 units mL–1 G6PDH, and 30 µL of MilliQ water. The reaction was monitored at an absorbance of 600 nm for 2 min with a Biotek Epoch 2 spectrophotometer. The concentrations of pyridine nucleotides were quantified by comparing the rates to those generated from a known range (1–100 pmol) of authentic oxidized or reduced pyridine dinucleotide standard of NADH, NAD+, NADPH, and NADP+ that were simultaneously assayed with the floret extracts.
2.7 Determination of glutathione reductase, ascorbate peroxidase, and dehydroascorbate reductase activities in the florets of stored broccoli
The activities of GR, APX, and DHA reductase were assayed according to Nakano and Asada (1981) and Noctor et al. (2023) with some modifications. Frozen floret powder (i.e., 500 mg) of each EGT-treatment replicate was combined with 2.5 mL of enzyme-specific extraction buffer and homogenized with a mortar and pestle. GR was extracted with 0.1 M KH2PO4: K2HPO4 (pH 7.5) containing 0.5 mM EDTA. APX was extracted with 0.1 M KH2PO4: K2HPO4 (pH 7.0) containing 1 mM EDTA, 1 mM ascorbic acid, 0.5% (w/v) polyvinylpolypyrrolidone, and 20% (w/v) sorbitol. DHAR was extracted with 0.1 M KH2PO4: K2HPO4 (pH 7.0) containing 1 mM EDTA and 0.5% (w/v) polyvinylpolypyrrolidone. Each extraction buffer contained the following protease inhibitors: 1 mM 1,10-phenanthroline, 1 mM phenylmethanesulfonyl fluoride, 334 µM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 14 µM E-64, 5.25 µM leupeptin, 3 µM bestatin, and 1 µM pepstatin A. All homogenates were filtered through a single layer of Miracloth (Fisher Scientific Canada), and then centrifuged at 13,000g and 4°C for 10 min. Thereafter, a 100 µL aliquot of the clarified extract was desalted on a 0.5 mL Sephadex G-25 spin column (Scouten and Elhardt, 1988).
All enzyme assays were performed on a BioTek Epoch 2 spectrophotometer. To determine GR activity, a 200 µL assay consisted of 0.15 mM GSSG, 0.15 mM NADPH, and 20 µL of the desalted floret extract, in 0.1 M NaH2PO4 buffer (pH 7.5) containing 20 mM EDTA. The assay was initiated by the addition of the desalted extract, and the depletion of NADPH (molar extinction coefficient = 6.22 mM cm–1) was monitored at an absorbance of 340 nm for 5 min. APX and DHAR activity assays were performed in 1-mL quartz cuvettes. For APX activity determinations, each 1 mL assay consisted of 25 mM KH2PO4: K2HPO4 (pH 7.0), 0.2 mM ascorbic acid, 0.2 mM H2O2 and was initiated by the addition of 50 µL of the desalted extract. Ascorbic acid depletion was monitored at an absorbance of 290 nm (molar extinction coefficient = 2.8 mM cm–1) for 1 min. For DHAR activity determinations, each assay consisted of 0.2 mM DHA and 1 mM GSH in 50 mM KH2PO4: K2HPO4 (pH 7.0) and was initiated by the addition of 50 µL of the desalted floret extract in the same buffer. The ascorbic acid production was monitored at an absorbance of 265 nm (molar extinction coefficient = 14.7 mM cm–1) for 2.5 min.
2.8 Statistical analysis
R Program (RStudio 2022.07.1) was used for all statistical analyses (R Core Team, 2024). All datasets were verified for normality and homogeneity by Shapiro–Wilk test and Levene’s test, respectively. Where required data were root square or log transformed to fit assumptions of normality and homoscedasticity. Two-way ANOVA partitioned variance into fixed effects (EGT treatment concentration and storage time) and their interactions. Thereafter, for all cases including where interactions were significant (p < 0.05), treatment means were compared at each sampling time over the storage period using Duncan’s test. Similarly, for each EGT concentration treatment Duncan’s test was used to compare differences between means across the storage period.
3 Results and discussion
3.1 Ergothioneine uptake in postharvest stored broccoli
The bottom portion of the stalk corresponding to each broccoli head was partially submerged in a solution containing a fixed concentration of EGT for a 24-h period. This partial submergence approach was used for broccoli subjected to four separate EGT concentrations, specifically 0 µM, 20 µM, 100 µM and 500 µM. Thereafter the broccoli heads were stored at 10°C for up to 10 days. EGT was not detected in broccoli florets of the 0 µM (i.e., control) treatment (Table 1). EGT was detected in all broccoli florets of heads that were partially submerged in solutions of exogenous EGT, regardless of the original concentration that was supplied to the head stalk (Table 1). This fits with previous research demonstrating the accumulation of EGT in seeds of plants that form symbiotic relationships with EGT-accumulating mycorrhizal fungi (Carrara et al., 2023). Similarly, EGT is detected in mustard seedlings grown hydroponically with low concentrations of this antioxidant (Kataoka et al., 2025). Overall, this implies that any absorbed EGT was translocated upwards into the broccoli florets following uptake by the partially immersed stalk. At the end of the 24-h partial submergence period (i.e., day 0 of storage) EGT was most prevalent in broccoli treated with 500 µM EGT where concentrations ranged from 3.3 to 7.2 µmol kg FW–1 (Table 1). For each EGT treatment, the EGT concentrations within the florets were not significantly different over the 10-day storage period. This contrasts with previous research on EGT-treated baby arugula whereby a 35% loss of the absorbed EGT occurred within 3 days of storage at 10°C (Sivakumar and Bozzo, 2024). Throughout the storage period, the EGT concentration detected in the florets of broccoli heads submerged in 500 µM EGT were 6.2 to 10.2-fold and 82 to 119-fold more than the concentrations detected in the 100 µM EGT-treated broccoli and 20 µM EGT-treated broccoli, respectively. This maximal amount of EGT detected in 100 µM EGT-treated broccoli is approximately 90% lower than the EGT detected in baby arugula leaves immersed in 100 µM EGT (Sivakumar and Bozzo, 2024). As the arugula leaves in the previous study were not rinsed after exposure to EGT, the possibility remains that some of the detected EGT could have been that adhered to the leaf surface.
Table 1. Ergothioneine (EGT) concentrations in the florets of broccoli heads stored at 10°C for 10 days following partial submergence in varying concentrations of EGT.
Several in vitro studies have established hercynine as the ultimate oxidation product of EGT (Ando and Morimitsu, 2021; Servillo et al., 2015). These same studies identified EGT disulfide, EGT sulfenic acid, EGT sulfinic acid, and EGT sulfonic acid as intermediate products of EGT oxidation. Moreover, hercynine was detected in beverages (e.g., teas) following derivatization with diethylpyrocarbonate and HPLC-ESI-MS/MS analysis (Sotgia et al., 2018). Although we can’t exclude the possibility that hercynine also occurs in EGT-treated broccoli, this may be unlikely given that the absorbed EGT concentrations were little altered with storage period. Alternatively, there may be a possibility that any oxidized EGT within the broccoli florets was recycled back to EGT. In fact, there is evidence that EGT-disulfide can be converted to EGT by mammalian GR and selenium-dependent thioredoxin reductase (Jenny et al., 2022). Similar phenomena have not been described for plant reductases.
3.2 Impact of exogenous ergothioneine on senescence and chlorophyll status of stored broccoli
The fresh biomass of broccoli heads was not altered during the storage period, regardless of the EGT treatment and concentration supplied prior to storage (Supplementary Figure S1). The color of broccoli is a primary criterion for consumer acceptance (Pellegrino et al., 2019). A visual assessment of the stored broccoli heads revealed less floret yellowing in broccoli treated with 20 µM EGT relative to all other treatments (Figure 2). Broccoli floret yellowing is due to chlorophyll degradation, which is most pronounced with temperature abuse conditions (Luo et al., 2020; Gao et al., 2018; Ahlawat et al., 2022). On day 0 of storage, the total chlorophyll concentration in the florets across all EGT-treatments was approximately 150 mg kg FW–1 (Figure 2). Our study revealed the florets had three times more chlorophyll a than chlorophyll b. This ratio is comparable to other broccoli studies (Costa et al., 2006; Wang et al., 2024c). Chlorophyll b decreased during 10°C storage but was not impacted by the pre-storage EGT applications. Losses of chlorophyll a and total chlorophyll were apparent by day 3 of storage, but not in the no EGT and 20 µM EGT treatments. Like the EGT-free broccoli, chlorophyll losses of 52% to 63% occurred in broccoli supplied with 100 µM and 500 µM EGT prior to storage. Chlorophyll degradation was less than the 90% loss described for broccoli stored at 20°C for 5 days (Costa et al., 2006). In addition, the loss of chlorophyll in our study was more than the 30% loss that was described for broccoli stored at 4°C for 25 days (Wang et al., 2024c). The florets of broccoli treated with 20 µM EGT had 29% more total chlorophyll than the control by day 10 of storage. This could be due to reduced degradation by chlorophyll catabolic enzymes such as chlorophyll reductase (Zhu et al., 2017). Generally, chlorophyll catabolism gene expression is elevated in senescing broccoli (Chen et al., 2008; Luo et al., 2019). The possibility remains that less chlorophyll catabolism gene expression occurred in broccoli submerged in 20 µM EGT relative to the other treatments. Broccoli yellowing overlaps with dismantling of the chloroplast by way of senescence-associated vacuoles (Bárcena et al., 2020). Moreover, postharvest senescence of Chinese flowering cabbage overlaps with increased expression of ROS biosynthesis genes (i.e., respiratory burst oxidase homologues) and ROS accumulation (Fan et al., 2018).
Figure 2. Dynamics of broccoli senescence and alterations in chlorophyll composition during postharvest storage of broccoli. Broccoli heads were partially submerged in aqueous solutions containing 0, 20, 100 or 500 µM ergothioneine (EGT) and then stored at 10°C for 10 days. Representative images of broccoli heads (A, B) following partial submergence in various EGT solutions and subsequent storage at 10°C for 10 days. Scale bar in each photo = 5 cm. (C) Storage duration related alterations in the concentrations of chlorophyll a, chlorophyll b and total chlorophyll in florets of broccoli treated with 0 µM (No EGT), 20 µM EGT, 100 µM EGT and 500 µM EGT. All chlorophyll concentrations are expressed on a fresh weight (FW) basis. Each datum represents the mean ± standard error of three independent experimental replicates. Within each chlorophyll plot, uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within each treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
3.3 Impact of exogenous ergothioneine on the dynamics of H2O2 production in broccoli during postharvest storage
Broccoli undergoes oxidative stress during postharvest senescence, which includes the accumulation of ROS (Li et al., 2014; Raseetha et al., 2013). H2O2 is the most stable ROS that occur in plants relative to its more reactive counterparts (e.g., superoxide) (Smirnoff and Arnaud, 2019). In addition, H2O2 is readily detected in plant tissue extracts (Chakraborty et al., 2016). The Amplex™ Red colorimetric assay revealed that the concentrations of H2O2 increased in the florets by as much as 7.8-fold throughout the storage period and peaked on day 10 (Figure 3). By the end of the storage period, broccoli treated with 20 µM EGT had 19% to 39% less H2O2 than all other treatments. The magnitude of control of H2O2 accumulation by 20 µM EGT is consistent with that exerted by the food additive diacetyl (i.e., 2,3-butanedione; Li et al., 2022). Given that there is no established evidence for the detoxification of H2O2 by EGT, the limited accumulation of H2O2 in 20 µM EGT-treated broccoli is likely due to the enhanced operation of the ascorbate-GSH recycling pathway (as discussed in section 3.5). Conversely, the increased accumulation of H2O2 in the florets of broccoli treated with 100 µM and 500 µM EGT may be a consequence of decreased activities of one or more ascorbate-GSH recycling pathway enzymes. The buildup of H2O2 may also be due to increased chelation of metal ions such as Cu2+ and Fe2+. A rationale for the latter is based on evidence that EGT complexes with metals such as Cu2+ (Motohashi et al., 1974; De Luna et al., 2013). Cu2+ and Fe2+ are known to promote the conversion of H2O2 into more reactive radicals. For example, excess copper in plants can form a hydroxyl radical from H2O2 via the Haber-Weiss cycle (Wang et al., 2024b). Moreover, it is known that metals complexed by EGT limits ROS-mediated damage of DNA (Zhu et al., 2011).
Figure 3. Alterations in hydrogen peroxide (H2O2) concentrations in the florets of broccoli following partial submergence in exogenous ergothioneine (EGT) solutions of various concentrations followed by storage at 10°C for 10 days. The four respective pre-storage EGT treatments included 0 µM (No EGT), 20 µM EGT, 100 µM EGT and 500 µM EGT. All H2O2 concentrations are expressed on a fresh weight (FW) basis. Each datum represents the mean ± standard error of three independent experimental replicates. Uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within each treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
3.4 Impact of exogenous ergothioneine on the pyridine dinucleotide profiles of stored broccoli
NADPH is required as a cofactor for many metabolic processes in plants, including the major ROS-generating enzyme NADPH-dependent oxidase activity, as well as the electron donor-driving GR activity in the ascorbate-GSH recycling pathway (Aghdam et al., 2020). We monitored shifts in the profiles of oxidized and reduced forms of phosphorylated pyridine dinucleotides in the broccoli florets as a function of storage period (Figure 4). NADPH concentrations within the florets were stable during storage. The sole exception was that the NADPH pool decreased by a third in 500 µM EGT-treated broccoli by day 7 of storage. The loss of NADPH in the 500 µM EGT-treated broccoli may be due in part to increased activity of NADPH oxidase, which produces superoxide. Although not assessed here, a coordinated increase in superoxide dismutase activity could have elevated H2O2 production in the 500 µM EGT-treated broccoli (Figure 3). For all other treatments, the stability of NADPH within the stored broccoli fits with the relatively unchanged concentrations of NADPH and NADP+ in whole melon fruit (Cucumis melo L. var. reticulatus Nand.) during storage at 15°C for 12 days (Wu et al., 2019). These metabolic trends were associated with minor changes in the NADPH oxidase activity within the melon fruit. Shigenaga et al. (2005) established that pre-stored broccoli florets of the ‘Erude’ cultivar contain approximately 24 µmol NADPH kg FW–1. This is nearly double the pre-storage concentration of NADPH in the ‘Imperial’ broccoli investigated in our study. More importantly, the Shigenaga et al. study found NADPH was unchanged in broccoli stored at 15°C for 6 days, whereas NADPH increased by approximately 70% in broccoli that were exposed to 50°C for 2 h before storage. With exception of the 500 µM EGT-treated broccoli, the homeostatic balance of NADPH may also be due in part to a restriction in its production by the oxidative pentose phosphate pathway. Decreased activities of the oxidative pentose phosphate pathway enzymes glucose 6-phoshpate dehydrogenase and 6-phosphogluconate dehydrogenase occur in senescing Chinese flowering cabbage during storage at 15°C (Wang et al., 2024a). It is worth noting that this same study found these activities are unchanged when senescence is controlled by the synthetic cytokinin 6-benzylaminopurine. It is worth noting that this pathway is dependent on the availability of carbon skeletons, but it is well established that concentrations of soluble sugars including sucrose decrease with postharvest senescence of broccoli (Hasperué et al., 2015).
Figure 4. Phosphorylated pyridine nucleotide redox profiles in the florets of broccoli following partial submergence in exogenous ergothioneine (EGT) solutions of various concentrations followed by storage at 10°C for 10 days. The four respective pre-storage EGT treatments included 0 µM (No EGT), 20 µM EGT, 100 µM EGT and 500 µM EGT. All NADPH and NADP+ concentrations are expressed on a fresh weight (FW) basis. Each datum represents the mean ± standard error of three independent experimental replicates. Uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within each treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
NADP+ concentrations within the stored broccoli declined by 37–57% over the storage period. The largest decreases were apparent in the no EGT-treated and 20 µM EGT-treated broccoli. By day 10 of storage, the NADPH: NADP+ ratio was as much as 52% smaller in the 100 and 500 µM EGT treatments than the other treatments. This redox ratio tended to remain relatively stable throughout the storage period in the broccoli florets of all four treatments. Overall, the lack of any sustained accumulation of phosphorylated pyridine dinucleotides including the loss of NADP+ could be due to alterations in NAD kinase activity. NAD kinase catalyzes the primary step promoting the phosphorylation of NAD+ to NADP+ in plants. Decreased NAD kinase activity and hence NADP+ concentrations occur with senescence of horticulture crops and are increased with treatments that delay senescence (Aghdam et al., 2020).
We also investigated the influence of the pre-storage exogenous EGT treatments on the shifts in the non-phosphorylated pyridine dinucleotide redox status within the florets of the stored broccoli heads. The finite concentrations of NADH increased 54% with storage period in broccoli treated with 20 µM EGT but not in the other treatments (Figure 5). To a similar extent, Wang et al. (2020) found a greater increase in NADH concentrations in controlled atmosphere-stored broccoli after they were soaked in a solution of exogenous GSH relative to those soaked in water. The NAD+ concentration declined by a similar proportion in this treatment over the 10-day storage period, although the decline was apparent 3 days earlier. Similar changes in the NAD+ concentration were apparent in the broccoli florets of the remaining treatments, although this change was not apparent until day 10 in the 100 µM EGT treatment. Overall, the NADH: NAD+ ratio increased 178% to 252% with storage, although there was no difference between the broccoli of the four different pre-storage treatments. Similar shifts in the profiles of the reduced and oxidized forms of non-phosphorylated pyridine dinucleotides have been described for controlled atmosphere-stored broccoli (Wang et al., 2020). It is likely that losses of soluble sugars required for aerobic respiration would limit NADH production from glycolysis and the tricarboxylic acid cycle. In fact, a decrease in ATP concentration and tricarboxylic acid cycle enzyme activities occur in broccoli during storage at 10°C, including in heads stored in refrigerated air (Li et al., 2016; Wang et al., 2020). The depletion of NAD+ in the broccoli florets may be due to their use by poly(ADP-ribose) polymerases (PARPs). PARPs are involved in the ADP-ribosylation of proteins during senescence (Hashida et al., 2009). A recent study by Aghdam and Alikhani-Koupaei (2021) revealed that strawberry senescence is reduced with the prepropeptide phytosulfokine α, and this was associated with the accumulation of both NAD+ and NADH, and reduced expression of PARP1 during storage at 4°C for 18 days.
Figure 5. Non-phosphorylated pyridine nucleotide redox profiles in the florets of broccoli following partial submergence in exogenous ergothioneine (EGT) solutions of various concentrations followed by storage at 10°C for 10 days. The four respective pre-storage EGT treatments included 0 µM (No EGT), 20 µM EGT, 100 µM EGT and 500 µM EGT. All NADH and NAD+ concentrations are expressed on a fresh weight (FW) basis. Each datum represents the mean ± standard error of three independent experimental replicates. Uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within each treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
3.5 Impact of exogenous ergothioneine on metabolic alterations of the ascorbate-glutathione recycling pathway in stored broccoli
The redox states of ascorbate and glutathione were monitored in the florets to assess whether the accumulation of H2O2 in the stored broccoli was associated with shifts in the operation of the ascorbate-GSH recycling pathway (Figures 6, 7). Ascorbate was stable between day 0 and day 3 of storage, regardless of the exogenous EGT concentration applied prior to storage (Figure 6) but dropped 33% to 46% thereafter. This magnitude of ascorbate degradation is less than the approximate 90% loss described for broccoli stored at 20°C (Mori et al., 2009; Nishikawa et al., 2003). Recent research has shown that ascorbate is 20% greater in baby arugula immersed in an EGT solution for 30 min and then stored at 4°C for 10 days as compared to non-antioxidant-treated leaves (Sivakumar and Bozzo, 2024). It is worth mentioning this effect was not sustained with prolonged storage thereafter nor for baby arugula stored at 10°C. Similarly, the application of exogenous EGT had little impact on the loss of ascorbate in the florets of the broccoli investigated in our study. This contrasts with the improved preservation of ascorbate and GSH in broccoli florets that were cut in half and then immersed in a 3.25 mM GSH solution for 5 min, although there was still a loss of both with storage (Jiao et al., 2025). Interestingly, the exogenous EGT application influenced the dynamics of DHA accumulation in the broccoli florets with storage. A 50% drop in the DHA concentration occurred in florets of the 20 µM EGT treatment by day 10 of storage. By the end of the storage period broccoli treated with 100 µM and 500 µM EGT had 1.1-fold more DHA in their florets as compared to the 20 µM EGT treatment. It is worth noting that the DHA concentration in the EGT-free broccoli and the 20 µM EGT-treated broccoli were comparable by the end of the storage period. Moreover, at this same time the ascorbate redox status (i.e., ratio of ascorbate to DHA) in the 20 µM EGT-treated broccoli was as much as 1.4-fold greater than all other EGT treatments. This concentration-dependent impact of exogenous EGT on ascorbate redox status of the stored broccoli is likely a consequence of the varying impact of these partial submergence treatments on the enzyme activities of the ascorbate-GSH recycling pathway as discussed below.
Figure 6. Ascorbate metabolite redox profiles in the florets of broccoli following partial submergence in exogenous ergothioneine (EGT) solutions of various concentrations followed by storage at 10°C for 10 days. The four respective pre-storage EGT treatments included 0 µM (No EGT), 20 µM EGT, 100 µM EGT and 500 µM EGT. All concentrations of the reduced form of ascorbate and its oxidized form dehydroascorbate are expressed on a fresh weight (FW) basis. Each datum represents the mean ± standard error of three independent experimental replicates. Uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within each treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
Figure 7. Glutathione metabolite redox profiles in the florets of broccoli following partial submergence in exogenous ergothioneine (EGT) solutions of various concentrations followed by storage at 10°C for 10 days. The four respective pre-storage EGT treatments included 0 µM (No EGT), 20 µM EGT, 100 µM EGT and 500 µM EGT. All concentrations of the reduced form of glutathione and its oxidized form glutathione disulfide are expressed on a fresh weight (FW) basis. Each datum represents the mean ± standard error of three independent experimental replicates. Uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within each treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
On day 0 of storage, a 40% greater concentration of GSH was present in the florets of the 20 µM EGT-treated broccoli relative to the control (Figure 7). This mirrors the elevated levels of GSH on day 0 of storage in fresh-cut broccoli florets immersed in exogenous GSH relative to broccoli immersed in water alone (Jiao et al., 2025). In broccoli sampled after day 0, we observed 95% and 62% spikes in the GSH concentration within the control and 20 µM EGT treatment, respectively. There was no such change in the remaining treatments. The GSH pool within broccoli treated with 20 µM EGT was 41% to 63% greater than that of the other two EGT treatments on days 3 and 7 of the storage period. GSH returned to its original concentration by day 10 of storage in the florets of the 20 µM EGT-treated broccoli and the EGT-free broccoli (i.e., control). This transient increase in GSH concentration in the 20 µM EGT-treated broccoli matches a similar spike in the GSH profiles of broccoli florets treated with the carotenoid cleavage product β-ionone and then stored at 15°C (Zhang et al., 2025). That study revealed that the accumulation of GSH in response to β-ionone coincided with increased gene expression of GR and the GSH biosynthesis enzymes γ-glutamylcysteine synthetase and glutathione synthetase relative to untreated broccoli. The upregulation of GSH biosynthesis gene expression is not likely associated with the GSH accumulation dynamics in the 20 µM EGT-treated broccoli as all treatments were held in the dark over the whole storage period. In fact, there is evidence that GSH biosynthesis is diurnally regulated in the leaves of oilseed rape (Brassica napus) plants, a botanical relative of broccoli (Hornbacher et al., 2019). The more likely mechanism responsible for the GSH spike in the 20 µM EGT-treated broccoli is the reduction of GSSG via the ascorbate-GSH recycling pathway.
In our study, it is worth noting that there was no transient fluctuation in the GSH pool with storage period in the florets of broccoli treated with 100 or 500 µM EGT. For these two EGT treatments, GSH concentrations declined 46% to 49% over the 10-day storage period. These losses were less than the approximate 80% loss of total glutathione in broccoli florets held at 23°C for 6 days (Raseetha et al., 2013). GSSG levels fluctuated with storage but there was little effect of the pre-storage exposure to exogenous EGT, regardless of the concentration supplied. A 70% spike in the glutathione redox status (i.e., ratio of GSH to GSSG) was apparent in broccoli treated with 20 µM EGT by day 7 of storage, but this alteration was transient. A similar phenomenon was apparent for the glutathione redox status of EGT-free broccoli, whereas there was little change over the storage period in the other two EGT treatments. By the end of the 10-day storage period the glutathione redox status approximated 2 in broccoli treated with 20 µM EGT and was as much as 88% greater than all other treatments.
The impact of exogenous EGT on shifts in the enzymatic activities of the ascorbate-GSH recycling pathway enzymes. This was done to assess whether the concentration dependent effects of exogenous EGT on the stored broccoli were correlated to changes in ascorbate redox and glutathione redox states (Figure 8). Previous research has established that marked reductions of GR, APX and DHA reductase activities occur within senescing broccoli during temperature abuse storage (Shigenaga et al., 2005; Mori et al., 2009; Raseetha et al., 2013). GR activity within the florets of the 20 µM EGT-treated broccoli was consistently greater than that of all other treatments over the 10-day storage period. GR activity spiked 10% to 19% on day 3 but declined thereafter for all four treatments. This contrasts with previous research by Jiao et al. (2025) where GR activity remained unchanged over the first 2 days of storage at 10°C and declined thereafter. It is worth noting that study did not monitor these biochemical changes in the stored broccoli after day 3. APX activity increased by 120% to 129% over the 10-day storage period. Over the first 7 days of storage, floret APX activity of the 20 µM EGT-treated broccoli was 13% to 16% less than the control broccoli, and 22% to 36% less than the other two EGT treatments. Shigenaga et al. (2005) reported a 50% decrease in DHA reductase activity of broccoli florets within heads held at 15°C for 6 days. Similarly, we observed a decline in DHA reductase activity with storage. This decline approximated 20% in the florets of the broccoli treated without and with 20 µM EGT but approximated 30% in the other EGT treatments. On day 7 of storage, the DHA reductase activity was up to 24% greater in the 20 µM EGT-treated broccoli relative to the other three treatments. Zhang et al. (2023a) determined that Chinese cabbage cultivated with exogenous EGT contained more root mass as well as altered expression of genes associated with the phenylpropanoid pathway and ascorbate metabolism, although the precise ascorbate metabolism gene changes were not elucidated. Nonetheless, it is known that gene expression of ascorbate-GSH pathway enzymes (e.g., DHA reductase) is downregulated in broccoli sealed in polyethylene bags and held at 20°C for 4 days (Zhang et al., 2023b). This same study found that the decrease in abundance of these transcripts was not as dramatically altered in broccoli treated with 2 mM H2O2. The possibility remains that the 20 µM EGT partial submergence treatment maintains the transcription of ascorbate-GSH recycling pathway genes in stored broccoli, whereas gene expression for this pathway may be downregulated with the other pre-storage treatments used in this study. A transcriptomics and/or proteomics analysis can elucidate the key genes and proteins involved in postharvest senescence of broccoli and establish the regulatory networks responsive to exogenous EGT, including those that impact senescence.
Figure 8. Impact of partial submergence of broccoli heads in exogenous ergothioneine (EGT) on the floret activities of ascorbate-glutathione recycling pathway enzymes during storage at 10°C for 10 days. Alterations in the specific activities of glutathione reductase, ascorbate peroxidase and dehydroascorbate reductase are expressed on a nmol min–1 mg protein–1 basis. Each datum represents the mean ± standard error of three independent experimental replicates. Uppercase letters denote statistical comparisons across all four treatments at each storage period sampling day. Lowercase letters denote statistical comparisons within a treatment across the 10-day postharvest storage period. In either case, shared letters indicate no differences between the means at p < 0.05.
4 Conclusions
The study tested the impact of pre-storage exposure to solutions of varying concentrations of the mushroom-derived antioxidant EGT on the quality and oxidative stress metabolism of stored broccoli heads. The antioxidant was delivered by partial submerging the broccoli in EGT solutions of varying concentrations. This strategy was chosen to minimize the wetting of the vegetable and prolonged drying thereafter. HPLC-MS/MS analysis revealed the EGT was efficiently acquired from the submergence solution and concentrated within the florets. The study determined that broccoli treated with the low concentration of 20 µM EGT delayed the loss of chlorophyll, limited H2O2 accumulation and better preserved the glutathione and ascorbate redox status of the florets relative to all other treatments, including broccoli treated with greater EGT concentrations. The 20 µM EGT treatment is a feasible strategy that broccoli farming operations and/or distribution centers can use to offset the development of advanced senescence due to ROS accumulation that can occur during transport, distribution or at the retail sector when the vegetable is exposed to temperature abuse.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Author contributions
YY: Formal analysis, Methodology, Writing – review & editing, Investigation, Writing – original draft. GGB: Formal analysis, Methodology, Writing – review & editing, Conceptualization, Funding acquisition, Project administration, Supervision.
Funding
The author(s) declared that financial support was received for this work and/or its publication. We gratefully acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (grant number ALLRP 560281-20) and an NSERC Postdoctoral Fellowship awarded to YY. Additional research funding was provided by the Ontario Ministry of Agriculture Food, Agribusiness (grant number UG-T1-2022-101692), as well as grant-in-aid research support from Mushrooms Canada, Berry Growers of Ontario, and the Fresh Vegetable Growers of Ontario.
Acknowledgments
The authors are grateful to Dyanne Brewer and Armen Charchoglyan at the mass spectrometry facility within the University of Guelph’s Advanced Analysis Centre for technical assistance with the HPLC-MS/MS analysis.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fhort.2025.1727923/full#supplementary-material
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Keywords: ascorbate, broccoli, ergothioneine, glutathione, postharvest, senescence
Citation: Yan Y and Bozzo GG (2026) Influence of partial submergence in exogenous ergothioneine on senescence and oxidative stress metabolism of broccoli during temperature abuse storage. Front. Hortic. 4:1727923. doi: 10.3389/fhort.2025.1727923
Received: 18 October 2025; Accepted: 01 December 2025; Revised: 29 November 2025;
Published: 15 January 2026.
Edited by:
Marwa Moumni, Marche Polytechnic University, ItalyReviewed by:
Simone Piancatelli, Marche Polytechnic University, ItalySarah M. Makau, University of Pretoria, South Africa
Copyright © 2026 Yan and Bozzo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Gale G. Bozzo, Z2JvenpvQHVvZ3VlbHBoLmNh