24-epibrassinolide alleviates postharvest yellowing of broccoli via improving its antioxidant capacity

Huixin Fang, Qian Zhou, Shunchang Cheng, Xin Zhou, Baodong Wei, Yingbo Zhao, Shujuan Ji *

Abstract

Postharvest crop yellowing is a major concern in the broccoli industry. The effect and underlying mechanisms of 24-epibrassinolide (EBR) treatment on yellowing in postharvest broccoli were investigated. Treatment with 2 µM EBR markedly inhibited the increase of the yellowing index and L* values, causing higher retention of the metric hue angle and chlorophyll content compared to the control. Treatment also alleviated oxidative damage by preventing the accumulation of malondialdehyde and superoxide anion (O2•− ). The ascorbic acid content of broccoli reached its lowest value at the end of its shelf life, whereas that of the treated sample was obviously higher than the control. Moreover, treated broccoli exhibited higher superoxide dismutase, ascorbate peroxidase, and phenylalanine ammonia-lyase activities. Multivariate statistical analysis further demonstrated the effective enhancement of EBR treatment on antioxidant enzymes. These results indicate that exogenous application of EBR ameliorates postharvest yellowing by improving the antioxidant capacity of broccoli.

Keywords:
Chlorophyll
Brassica oleracea
Postharvest storage Oxidative stress
Multivariate analysis

1. Introduction

Broccoli (Brassica oleracea L. var. italica) is an edible vegetable known for its health-promoting phytochemicals, such as vitamins, phenolic compounds, glucosinolates, and sulforaphane nitrile, and widely cultivated worldwide due to its consumer popularity (Fernandez- ´ Leon, ´ Fernandez-Le´ on, ´ Lozano, Ayuso, & Gonzalez-G´ omez, ´ 2013). However, the harvested broccoli bulbs, composed of dense clusters of buds, are quickly metabolized and prone to turn from green to yellow, which is accompanied by changes in nutritional composition and leads to the deterioration of product quality. Hence, it is of great practical significance to explore conservation technology.
Recently, researchers have committed to understanding the mechanism of broccoli yellowing and exploring effective associated control techniques. For instance, our previous study has shown that the green- to-yellow conversion process in broccoli occurs at the bud stage, before blooming, and gradually progresses from the base of the buds to the top, indicating that this phenomenon is mainly caused by the change in calyx pigments (Fang et al., 2020). Another emerging study has revealed that yellowing in postharvest tea was directly connected with the content of various chlorophyll derivatives (Yu et al., 2019). Moreover, Luo et al. (2019) found that chlorophyll content decreases during the early stage of the broccoli fading from green to yellow, whereas a significant rise in carotenoid content was detected as the degree of yellowing intensified. Thus, chlorophyll degradation is likely the principal cause of the color change from green to yellow in broccoli. Research on the structure of different chlorophyll catabolites in senescent leaves found that pheophorbide a oxygenase (PAO) catalyzes the opening of a porphyrin macrocycle ring of pheophorbide a and further promotes chlorophyll degradation (Hortensteiner, ¨ & Krautler, 2011¨ ). Intriguingly, PAO showed specificity for pheophorbide a, but was inhibited by pheophorbide b, indicating that conversion of chlorophyll b to chlorophyll a is a prerequisite for further degradation via PAO (Hortensteiner, 2012¨ ). Furthermore, the protein encoded by chlorophyll a oxygenase (CAO) cloned from Arabidopsis thaliana has been demonstrated to promote the oxidation of chlorophyll a to form chlorophyll b (Oster et al., 2000). The reduction of chlorophyll b, catalyzed by the chlorophyll b reductase and encoded by the non-yellow coloring 1 (NYC1) gene, is a key step in regulating senescence (Kusaba et al., 2007). Chlorophyll degradation is known to be induced by many factors. For instance, ethylene has been reported to regulate chlorophyll degradation and promote fruit ripening and senescence, thereby affecting crop quality (Knee, Tsantili, & Hatfield, 2010).
Reactive oxygen species (ROS) are by-products of various metabolic pathways in plants and have been identified as secondary messengers in phytohormone responses (Kwak et al., 2006). Plants themselves produce a certain amount of ROS over the course of normal physiological metabolism. In addition, ROS scavenging systems exist in plants, including antioxidant enzymes and antioxidants, which can counteract the overproduction of ROS and maintain ROS homeostasis (Apel & Hirt, 2004). However, the equilibrium between the generation and elimination of ROS might be disturbed by a variety of adverse stress factors, such as senescence, low temperatures, salinity, drought, and heavy metals, which provoke an increase in ROS production and a weakening of ROS scavenging capacity, thus leading to the accumulation of ROS. Excessive ROS triggered by either biotic or abiotic stresses is pernicious, accelerating the loss of chlorophyll and leading to senescence (Li et al., 2014). It also affects numerous cellular functions, causing inevitable damage to carbohydrates, proteins, lipids, and DNA, and eventually resulting in oxidative stress (Gill & Tuteja, 2010). Yellowing is an intuitive sign of senescence in broccoli, while the antioxidant system of plants gradually enhanced to resist the oxidative stress as senescence progresses. Therefore, preventing the excessive synthesis of ROS and improving scavenging capacity may be a strategy to delay the postharvest yellowing in broccoli.
Under physiological steady state conditions, antioxidant defense systems act synergistically to scavenge ROS and protect plant cells from oxidative damage. Superoxide dismutase (SOD; EC 1.15.1.1) is the first defense enzyme involved in the scavenging of intracellular superoxide anion-free radicals (O2•− ) and catalyzes them into molecular oxygen and hydrogen peroxide (H2O2). Catalase (CAT; EC 1.11.1.6), peroxidase (POD; EC 1.11.1.7), and ascorbate peroxidase (APX; EC 1.11.1.11) are major antioxidant enzymes in plants, as they have the ability to break down excess H2O2 and play a crucial role in the oxy-radical detoxification process (Tian, Qin, & Li, 2013). A higher quantity of antioxidant enzymes can contribute to protecting postharvest broccoli against the oxidative stress induced by ROS (Lemoine, Chaves, & Martínez, 2010). Furthermore, phenylalanine metabolism, which is the main pathway of phenol synthesis in plants, can be regulated as a defense mechanism to resist oxidative damage. Maintaining the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is beneficial to the accumulation of phenolic substances, and, hence, improves the antioxidant capacity of broccoli.
Brassinosteroids (BRs), a new class of steroid hormones ubiquitously found in plants, are recognized for their ability to regulate the postharvest quality of horticultural crops. It has been reported that BRs can ameliorate plant tolerance to abiotic stress by regulating the transcription of genes that encode protective proteins vital for stress resistance (Ahammed, Li, Liu, & Chen, 2020). BRs are active even at very low concentrations (10-8 M), but the use of analogues or the application of specific concentrations may result in different responses (Siddiqui, Ahmed, & Hayat, 2018). 24-epibrassinolide (EBR) is a bioactive BR with favorable safety and commercial availability. A recent study has highlighted the positive regulatory role of EBR in improving the defense mechanism of table grapes and activating antioxidant systems in response to ripening and aging (Asghari & Rezaei-Rad, 2018). Likewise, postharvest applications of EBR have been confirmed to effectively maintain the quality of peaches (Gao et al., 2016) and strawberries (Sun et al., 2019) by regulating their antioxidant systems. Moreover, Cai et al. (2019) reported that different concentrations of EBR had distinct effects on broccoli yellowing, and that a 2 µM EBR treatment effectively delayed the yellowing by inhibiting ethylene synthesis and chlorophyll degradation. Considering the impacts of ROS on plant ripening and senescence, as well as the regulatory effect of EBR on abiotic stress, research with a focus on oxidative stress may further explain the mechanisms by which EBR delays broccoli yellowing.
Based on a previous study about the dose–response of EBR treatment on broccoli yellowing conducted in our laboratory (Cai et al., 2019), here we verified the potential role of EBR and explored the underlying mechanism by which EBR delays broccoli yellowing, as related to oxidative stress. To do so, we observed visual changes in broccoli, evaluated O2•− levels and lipid peroxidation, and monitored the activity of antioxidant enzymes and antioxidant contents. Moreover, we performed a multivariate statistical analysis to further investigate the influence of EBR treatment on the antioxidant system of broccoli. This study offers novel insight into the effects of exogenous EBR in postharvest broccoli and unravels the corresponding mechanisms from the perspective of antioxidants, hence providing a theoretical basis to address the problem of postharvest yellowing in broccoli.

2. Materials and methods

2.1. Plant materials and treatment

Broccoli (Naihan-Youxiu) heads were harvested from a commercial plantation located in Jinzhou, Liaoning Province, China, and immediately transported to the laboratory in Shenyang Agricultural University, Liaoning Province, China, arriving within three hours postharvest. Samples of tight-head broccoli (9–13 cm in diameter) that were uniform in color, maturity, and size, without any disease or mechanically- induced wounds, were selected as experimental materials. The harvested broccoli heads were randomly divided into two groups containing 60 heads per group. One group was treated with 2 µM EBR (containing 2% EtOH), while the other was treated with distilled water (containing 2% EtOH) and served as control. Three biological replicates were performed for each treatment. Each head was evenly sprayed until liquid dripped from the surface. Afterwards, the treated samples were air-dried and placed in a plastic box lined with 0.03 mm thick polythene bags. The flower balls were arranged in a single layer to prevent crushing and stored at 10 ◦C ± 0.5 ◦C with 85% relative humidity for 10 d. Observation and a visual assessment of color were made every two days by random sampling from 20 heads. Florets were removed from the harvested broccoli heads on days 0, 2, 4, 6, 8, and 10, and were instantly frozen in liquid nitrogen and stored at − 80 ◦C for subsequent analysis of biochemical changes. The experimental design was completely randomized with three biological replicates.

2.2. Evaluation of phenotypic color

The appearance of broccoli was evaluated by observing the yellowing area of floret surfaces on the scale with reference to Luo et al. (2019). The results were calculated according to the following formula: Yellowing index (%) = Σ [(yellowing scale) × (number of broccoli at this scale)] / [(the maximum yellowing scale) × (total number of broccoli)] × 100.
A digital colorimeter (Chroma Meter CR-400, Konica Minolta, Tokyo, Japan) was used to determine the color parameters of broccoli. Six broccoli heads of each replicate were randomly selected, and parameters were measured three times at five different points on each broccoli head and expressed as L* (L*=0 corresponds to black; L*=100 corresponds to white), a* (negative indicates green and positive indicates red), and b* (negative indicates blue and positive indicates yellow). The metric hue angle (H◦) was calculated according to the CIE. Lab system, as H◦ = arc tan (b*/a* ).

2.3. Chlorophyll fluorescence and chlorophyll content analysis

High-resolution digital fluorescence images, values of non- photochemical quenching, which can be used to quantify the light protection capability of plants, maximum quantum yield (Fv/Fm), and fluorescence decline ratio (Rfd) were obtained noninvasively using an Open FluorCam7 (Photon Systems Instruments, Drasov, Czech Republic) with a CCD camera equipped with 720 by 560 pixels and 12-bit dynamic pixel resolution. Through optimization testing, we selected the optimal light saturation intensity (i.e., 2280 μE). Total chlorophyll content was determined according to Lichtenthaler (1987), and the contents of chlorophyll a and b were measured as described by Fang et al. (2020). There were three biological replicates per treatment, and the experiments were performed in triplicate.

2.4. O2•− and malondialdehyde (MDA) measurements

The generation rate of O2•− was measured according to Gao et al. (2016), with slight modifications. Broccoli tissue (1 g) was homogenized with 3 mL of pre-cooled phosphate buffered solution (pH 7.8) and centrifuged at 8000 × g for 10 min at 4 ◦C. Then, the supernatant (2 mL) was mixed with 0.5 mL of 50 mM potassium phosphate buffer and 0.1 mL of 10 mM hydroxylamine hydrochloride. After 20 min at 25 ◦C, 1 mL of 58 mM p-aminobenzene sulfonic acid and 1 mL of 7 mM 1-naphthylamine were added. The reaction mixture was shaken evenly and incubated at 30 ◦C for 30 min. Finally, the absorbance of the supernatant was measured at 530 nm, and the O2•− content was calculated using NaNO2 as a standard.
The determination of MDA content was performed using the methods of Meng and Min (2012), with slight modifications. Each broccoli sample (1 g) was ground in 5 mL of 10% (w/v) trichloroacetic acid, then centrifuged at 12 000 × g for 30 min at 4 ◦C. Afterwards, 2 mL of supernatant and 2 mL of 0.67% (w/v) thiobarbituric acid were added to the tube, and the mixture was held in boiling water for 15 min. After cooling down to room temperature, the mixture was centrifuged at 10 000 × g for 15 min. The absorbance of the supernatant was measured using a spectrophotometer (TU-1810 DSPC, Puxi Instrument Co., Beijing, China), and the MDA content was calculated with the following formula: Where A450, A532, and A600 are the absorbance values at 450, 532, and 600 nm, respectively, Vt and Vs are the total volume of the extract solution and volume of extract solution involved in the reaction, respectively, and m represents the mass of the sample.

2.5. Determination of enzyme activities

Each broccoli sample (1 g) was homogenized with 5 mL of pre-cooled 0.1 M phosphate buffer (pH 7.8) containing 5 mM dithiothreitol and 5% polyvinylpyrrolidone (PVP), then the mixture was transferred to a tube and swirled for 30 s. After 10 min of static extraction in an ice bath, the homogenates were centrifuged at 13 000 × g for 20 min. The obtained supernatant was used to measure enzyme activities. All steps of the extraction process were performed at 0–4 ◦C. SOD activity was measured using the method of Beyer and Fridovich (1987), based on the photoreduction of nitroblue tetrazolium (NBT) in the presence of riboflavin and methionine. One unit of SOD activity was defined as the amount of enzyme that resulted in 50% inhibition of the NBT photoreduction reaction. Absorbance was monitored at 450 nm. CAT activity was determined using the method of Hu et al. (2012), based on the enzyme- catalyzed decomposition of H2O2. Absorbance was monitored at 240 nm. One unit of CAT activity was defined as the amount of enzyme that catalyzed the decomposition of 1 mol of H2O2 per min. POD activity was determined using the method of Chu et al. (2018), with slight modifications. The reaction mixture contained 2.8 mL of 25 mM guaiacol, 0.2 mL of 0.5 M H2O2, and 0.5 mL of crude enzyme extract. One unit of POD activity was defined as the amount of enzyme that produced an increase in absorbance at 420 nm per min. APX activity was determined according to the method of Nakano and Asada (1981), based on the reduction in absorbance of ascorbic acid. One unit of APX activity was defined as the quantity of enzyme needed to cause a decrease in the absorbance at 290 nm per min. The values of the above-mentioned antioxidant enzyme activities were indicated as units per milligram of protein. Protein content of samples was measured according to the method described by Bradford (1976).
Finally, PAL activity was determined on the basis of a modified method (Assis et al., 2001). Each broccoli sample (1 g) was homogenized with 5 mL of 0.2 M borate buffer (pH 8.8) containing 2 mM EDTA, 5 mM β-mercaptoethanol, and 6 g PVP in an ice bath. The obtained homogenate was centrifuged at 13 000 × g for 20 min at 4 ◦C, and the supernatant was used to assay PAL enzyme activity. The reaction mixture contained 1 mL of 0.6 mM L-phenylalanine, 2 mL of 0.2 M borate buffer, and 0.5 mL of the supernatant in a total volume of 3.5 mL, and the reaction solution was placed in a water bath at a constant temperature of 37 ◦C for 60 min. The amount of PAL enzyme required for an absorbance change at 290 nm of 0.01 per min was defined as one unit, and data were expressed on a fresh weight basis as units per gram.

2.6. Measurement of ascorbic acid, total phenol contents, and 1,1- diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging rate

Ascorbic acid content was measured by using the 2,6-dichlorophenol-indophenol dye method described by Nath et al. (2011), and results were expressed on a fresh weight basis as milligrams per hectogram. The total phenol content was evaluated according to the method described by Hinneburg et al. (2006), with slight modifications. Each broccoli sample (2 g) was homogenized with 5 mL of methanol in an ice bath, and then centrifuged at 12 000 × g for 30 min at 4 ◦C. The supernatant (0.5 mL) was collected to measure the total phenol content. Thereafter, 1 mL of Folin-Ciocalteu reagent and 3 mL of 1 M sodium carbonate were added to the test tube, and the mixture was shaken gently. The total volume of the mixture was brought up to 10 mL with distilled water, after which the mixture was mixed thoroughly and kept at room temperature for 60 min. The absorbance was read at 760 nm, and the results were expressed as grams of gallic acid per kilogram of tissue.
DPPH assay was performed as described by Larrauri et al. (1998), with minor modifications. Each broccoli sample (1 g) was extracted with 50% ethanol and centrifuged at 12 000 × g for 10 min at 4 ◦C. In the presence of antioxidants, the intensity of the purple color of the DPPH solution declined, and the change in absorbance was measured at 517 nm. An ethanolic solution served as the control. The results were calculated using the following formula: DPPH radical scavenging rate (%) = 100- (absorbance of sample / absorbance of control) × 100.

2.7. Statistical analysis

All the data plotted in figures were presented as the mean ± standard deviation of three independent biological replicates, and were statistically analyzed with a one-way analysis of variance (ANOVA) using SPSS 20.0 software (IBM, Armonk, NY). Duncan’s multiple range test was used to compare the means, and significant differences were characterized at the 5% level. Differences at P < 0.05 were considered significant; differences at P < 0.01 were considered extremely significant. Simca-p11.5 statistical analysis software was used for principal component analysis (PCA) and partial least square regression (PLSR), and DPS 8.01 software was used for path analysis. Before each analysis, the data were standardized by z-score.

3. Results

3.1. Effects of exogenous EBR treatment on the color and related indicators of broccoli

Yellowing is the most visually apparent quality deterioration of postharvest broccoli. The broccoli in the control group began to turn yellow on day 6, whereas the samples treated with EBR maintained their initial dark green color at that time and showed slight yellowing two days later (Fig. 1A, B). Moreover, the yellowing index of EBR-treated broccoli was significantly lower than that of control broccoli during later shelf life (P < 0.01; Fig. 1C). Therefore, the EBR treatment effectively inhibited the occurrence and development of postharvest yellowing in broccoli.
To confirm the results of visual observation, samples were measured using a colorimeter. In both treatments, L* values increased and H◦ values continued to decline with the yellowing process (Supplementary Table). Compared to those of control samples, the L* values of EBR- treated broccoli were significantly lower, while the decline in H◦ values in the treated samples was retarded.
The color-coded images presented visible discrepancies between the two treatments on day 8 (Fig. 1D). The Fv/Fm values of the control samples decreased conspicuously from the 6th day of shelf life, while those of EBR-treated broccoli remained high (Fig. 1E). The Rfd values of both control and EBR-treated broccoli were characterized by an overall downward trend with the extension of shelf life. However, the decline rate and amplitude of the Rfd values of EBR-treated broccoli were much lower than those of the control samples.
A decline in chlorophyll a content was observed in both treatments from the 6th day (Fig. 1F). However, their chlorophyll b content exhibited a slight rise on day 2, and thereafter, it began to decline. Chlorophylls a and b in the control samples decreased by more than 60% until the end of the shelf life, and the EBR treatment slowed down their reduction. The upregulated expression of BoCAO intensified the conversion of chlorophyll a to chlorophyll b, which was then reconverted into chlorophyll a via BoNYC1 catalysis (Luo et al., 2019). In the early stage of the shelf life, the increase of chlorophyll b may be because of the ability of BoCAO to catalyze the synthesis of chlorophyll b, overcoming the degradation of chlorophyll b by BoNYC1.

3.2. Changes in O2•− and MDA content

A continuous accumulation of O2•− was observed in the control samples, reaching its highest level on day 6, while the O2•− production in EBR-treated broccoli was relatively stable and peaked two days later, at a value that was noticeably lower than that of the control (Fig. 2A). Over time, the MDA content in both the treated and control samples gradually increased (Fig. 2B). However, it was consistently lower in treated broccoli than in control samples, suggesting that the EBR treatment effectively relieved the oxidative damage of the product.

3.3. Changes in key antioxidant enzyme activity

The activities of SOD and APX in the control samples continuously decreased over the entire storage period (Fig. 3A, D), but the EBR treatment significantly delayed and lowered the amplitude of their reduction compared with that in control samples. Notably, CAT activity in the control sharply decreased during the first six days of shelf life, followed by an increase and a subsequent decline (Fig. 3B). Conversely, CAT activity in the treated samples slightly fluctuated, and was markedly higher than that in the control. The increase in POD activity detected in the treated samples was slower than that detected in the control, especially from the 4th day of shelf life onwards (Fig. 3C). Additionally, POD activity was lower in treated broccoli than in the control during the same period. Finally, PAL activity in the control fluctuated during the initial four days and continually decreased thereafter, while that in the treated samples remained high during the entire shelf life, despite some volatility (Fig. 3E).

3.4. Changes in ascorbic acid, total phenol contents, and DPPH radical scavenging rate

The ascorbic acid content in both EBR-treated and control broccoli decreased until the end of the shelf life (Fig. 4A). The ascorbic acid levels in treated broccoli were 64.6% and 92.8% higher than those in the control on days 8 and 10, respectively (P < 0.05). Notably, the decline range in EBR-treated samples was consistently smaller than that in controls. Total phenol content increased in the control with the extension of shelf life, peaking on day 8 and subsequently decreasing (Fig. 4B). In contrast, the total phenol content in the treated samples rose progressively, especially halfway through shelf life.
DPPH radical scavenging rate in the control samples exhibited a rapid rise during the first 8 days, followed by a decline (Fig. 4C). Notably, a higher increase in DPPH radical scavenging rate was detected in treated broccoli than in the control throughout shelf life. These results indicated that the accumulation of total phenol content in postharvest broccoli possibly contributed to DPPH radical scavenging. In conclusion, EBR treatment may effectively improve ROS scavenging capacity.

3.5. Multivariate statistical analysis

The cumulative contribution rate of the first two principal components (i.e., PC1 and PC2), which better explain the characteristics of the overall data, was 86.7% (Fig. 5A). Notably, chlorophyll, H◦, ascorbic acid, Fv/Fm, Rfd, SOD, and APX had high positive loadings on PC1, whereas the yellowing index, MDA, POD, and L* value had high negative loadings, which indicates that PC1 mainly reflected the changes in tissue color, senescence, and antioxidant capacity. Meanwhile, CAT, total phenol, and DPPH were the indicators with high positive loadings on PC2, while O2•− had a high negative loading, highlighting that PC2 reflected the production and scavenging ability of ROS in floret tissue. In addition, PC1 identified the samples of two treatments after six days of shelf life, and PC2 distinguished well between control and EBR-treated samples as a whole (Fig. 5B). Therefore, this implies that EBR had a significant impact on the changes in the above-mentioned indicators.
The correlation analysis illustrated the internal causes of the color changes in broccoli after the application of the EBR treatment (Fig. 5C). L* and H◦ were negatively and positively correlated with chlorophyll, respectively. The chlorophyll content had a significantly high correlation coefficient with enzyme activities of SOD, POD, and APX. Moreover, the yellowing index was significantly negatively correlated with SOD, APX and PAL activities, but positively correlated with POD activity. These results indicate that these enzymes may play a vital role in broccoli yellowing.
A PLSR model was established to further verify the involvement of antioxidant enzymes in broccoli yellowing. The yellowing index and chlorophyll values were selected as dependent variables (Y), and other indicators as independent variables (X). The yellowing index was remarkably positively correlated with MDA and POD, and noticeably negatively correlated with chlorophyll, SOD, and APX (Fig. 5D). These results suggested that broccoli yellowing was mainly affected by antioxidant enzymes, including SOD, APX, and POD, reflected by the yellowing index. They also indicated that with the aggravation of broccoli
Excessive ROS production could promote broccoli senescence, further manifested as yellowing, and the chlorophyll content was related to the color of broccoli. Therefore, path analysis was conducted with chlorophyll as the dependent variable and other indicators as independent variables, and the overall results are displayed in Table 1. The order of the direct path coefficients of antioxidant enzymes to chlorophyll was SOD > APX > PAL > POD > CAT, and the determinant coefficient of each independent variable to chlorophyll was 0.972, P = 0.002. Specifically, the direct path coefficients of SOD and APX to chlorophyll were both positive and large, with values of 0.448 and 0.304, respectively, and the indirect path coefficients of these two to chlorophyll through each other were also large. Moreover, the direct path coefficients of CAT, POD, and PAL were relatively small; comparatively, their indirect path coefficients to chlorophyll through SOD and APX were large. Our results suggest that these antioxidant enzymes play a synergistic role in broccoli yellowing.

4. Discussion

Broccoli is prone to yellowing during transportation and storage, resulting in the considerable reduction of its commercial value. In the present study, we found that yellowing symptoms were alleviated by the application of 2 µM EBR to postharvest broccoli, as illustrated by the lower yellowing index of the treated group in comparison with that of the control group. The lower amplitude of the rise in the L* value and the inhibition of the decrease in the H◦ value that were recorded with the treated samples further indicate that EBR treatment slows the color transition of broccoli from green to yellow. Furthermore, we observed that the chlorophyll content was characterized by a downward trend concomitant with the gradual loss of the green color over time. However, exogenously applied 2 µM EBR was effective in suppressing the decline in chlorophyll. It is worth noting that the impacts of different concentrations of EBR on broccoli have been reported to be discrepant, and, for instance, 20 μM EBR accelerates the degradation of chlorophyll (Cai et al., 2019).
Broccoli is still capable of photosynthesis after harvest. The Fv/Fm ratio is known to reflect the maximum quantum efficiency of photosystem II (PSII), and also is a crucial indicator of the degree of photoinhibition, that is, the higher the ratio, the lower the degree of photoinhibition. Excessive ROS can affect the transport of photosynthetic electrons and induce damage of the photosynthetic apparatus, but EBR counters this effect (Ahammed et al., 2013). In the current study, the Fv/Fm value in the control group decreased conspicuously after the 6th day of shelf life, suggesting that the degree of photoinhibition was significantly aggravated. This may be due to the inactivation of the PSII reaction center or structural damage, as well as the destruction of the energy dissipation mechanism by redundant ROS. In contrast, broccoli treated with EBR exhibited higher Fv/Fm values. Thus, EBR has the potential to alleviate photoinhibition.
Various stresses can impair the cell membrane system and cause the destruction of membrane integrity. For instance, MDA, the product of membrane lipid peroxidation, has been regarded as a marker of ROS- related oxidative damage. Lipid peroxidation accelerates the loss of chlorophyll in broccoli during postharvest storage, leading to quality deterioration (Zhuang et al., 1995). Furthermore, advanced senescence in broccoli has been reported alongside increased MDA levels, hinting at the possibility that lipid peroxidization causes membrane damage (Xu et al., 2012). As storage time increases, the cell membrane is increasingly vulnerable to oxidative damage. In the present study, the enhanced accumulation of O2•− , accompanied with increasing MDA levels, was observed during the process of broccoli yellowing, which further indicates that yellowing promotes oxidative stress. However, EBR treatment markedly impeded the production of O2•− and MDA, and the delayed yellowing observed in the treated samples may be attributed to the fact that EBR had a beneficial effect on mitigating lipid peroxidation.
Fruits and vegetables can defend themselves against abiotic stress and delay senescence through their antioxidant system. There are now multiple lines of evidence that antioxidant enzymes play a vital role in maintaining the quality of horticultural crops (Asghari & Rezaei-Rad, 2018; Chu et al., 2018; Hu et al., 2012). The effect of antioxidant enzymes has been reported to be crucial for preserving the green color of broccoli, and enhanced antioxidant enzyme activities may be a defense response to excess ROS (Toivonen & Sweeney, 1998). The results of the present study demonstrated that EBR treatment visibly retards broccoli yellowing and maintains higher activities of SOD, CAT, and APX. Additionally, the correlation and PLSR analyses further highlight that the yellowing index is significantly related to both SOD and APX. It has been reported that the POD activity in broccoli is suppressed after 6-benzylaminopurine treatment, which, in turn, postpones the degradation of chlorophyll (Xu et al., 2012). In the current study, the POD activity in control samples displayed a rising trend during yellowing, while the EBR treatment inhibited this increase. This result was consistent with the findings of Funamoto et al. (2002), who reported that a delay in chlorophyll degradation in heat-treated broccoli was strongly associated with lower POD activity. Furthermore, path analysis shows that the synergistic effect of antioxidant enzymes, such as SOD and APX, may be conducive to reducing oxidative damage. Given the above, our findings further indicate that antioxidant enzymes prominently contribute to the alleviation of broccoli yellowing.
Non-enzymatic antioxidant systems, such as ascorbic acid and phenolic compounds, have the function of scavenging ROS and protecting against oxidative damage. There is a controversial debate in the literature regarding the influence of ascorbic acid on the antioxidant capacity of fruits and vegetables (Prior et al., 1998; Guo et al., 2003; Liu et al., 2014). In this study, we found that the antioxidant capacity of broccoli may be more dependent on other antioxidant components than ascorbic acid. Senescence promoted the decrease of ascorbic acid content, making broccoli more prone to nutrient losses, whereas exogenous EBR prevented a decline in ascorbic acid content. Moreover, the total phenol content was significantly positively correlated with the DPPH radical scavenging rate, suggesting that total phenol had an important contribution to the antioxidant capacity of broccoli. A significantly higher total phenol content was detected in treated samples, which might be ascribed to the fact that EBR treatment enhanced PAL activity. Notably, the accumulation of phenols may serve the crucial function of responding to oxidative stress. Our findings aligned with those reported for peach fruit treated with exogenous EBR (Gao et al., 2016). Additionally, EBR-treated broccoli presented a higher DPPH radical scavenging rate compared with that in the control ones, indicating that exogenous EBR enhances the antioxidant capacity of broccoli. These findings suggest that EBR treatment participates in the induction of the antioxidant system and considerably offsets the adverse impacts of oxidative damage. Nonetheless, extensive research is still needed to gain a deeper insight into the function of EBR in regulating broccoli yellowing. In view of the excellent biological activity and safety of EBR, which does not have toxicological effects, the potential role of EBR in protecting broccoli against yellowing is of great interest.

5. Conclusion

In contrast to previous research on the impact of different concentrations of EBR on broccoli, this study revealed the positive effect and underlying mechanisms of 2 µM EBR treatment on delaying broccoli yellowing. Our data showed that EBR treatment prevented the increase in yellowing index and L* values, while sustaining higher H◦ values, chlorophyll concentration, and chlorophyll fluorescence parameters, all of which correspond to a better retention of the green color. The EBR treatment reduced oxidative stress by mitigating membrane lipid peroxidation, as reflected by lower MDA content, and had the potential to alleviate photoinhibition. In addition, it increased the activities of SOD, APX, and PAL and the accumulation of phenols, which indicates the significant role of EBR in enhancing the antioxidant capacity of broccoli. The results of multivariate statistical analysis further corroborated the potent enhancement impact of EBR treatment on antioxidant enzymes. Overall, we propose that EBR treatment may be a novel, effective, and feasible method to relieve broccoli yellowing by improving the antioxidant system of broccoli.

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