Regulation of pericarp browning in cold-stored litchi fruit using methyl jasmonate

2.1 Weight loss, pericarp browning (PB) and fruit decay

Fruits were weighed daily during storage and subsequently, the weight loss was calculated considering initial and final weight on each sampling day. PB was calculated using the browning intensity score explained by Kumar (2000). The number of fruit in each category was multiplied with the respective browning score and expressed in per cent (%). Decay loss was computed according to Ali et al. (2016) and expressed as a per cent (%).

2.2 Total soluble solids (TSS), titratable acidity (TA), and ascorbic acid (AA) content

The TSS (%) of juice extracted from litchi aril was estimated using a digital refractometer (Make-Hanna, Germany). To analyse TA, a diluted juice sample was titrated against NaOH solution (0.1 N) in the presence of phenolphthalein indicator. The recorded titre value was used for calculating TA and expressed as per cent (%) of citric acid (AOAC, 2000). For the determination of AA content, the fresh juice sample was diluted with 3% metaphosphoric acid (1:10) and 10 mL aliquot was titrated against the 2, 6-dichlorophenol indophenol dye till the appearance of pink colour. The results were expressed as mg L^-1 (Jones and Hughes, 1983).

2.3 Anthocyanin content

For the extraction of anthocyanin, 1 g of pericarp was taken in 9 mL methanol-HCL solution (85:15 v/v), then homogenised and kept overnight. The extract was centrifuged at 6000 × g and the optical density (OD) of the supernatant was recorded at 535 nm in a UV–Vis spectrophotometer (Dynamica HALO DB-20S, Australia). The total anthocyanin contents were calculated in terms of cyanidin 3- glucoside and expressed in mg kg⁻¹.

2.4 Total phenolics

Phenolic contents of pericarp were extracted in ethanol (80%) on a 1: 10 (w/v) ratio. The homogenised extract was centrifuged at 10000 × g for 20 min at 20 °C and the collected supernatant was used in total phenolics assay. The reagent mixture containing 100 μL pericarp extract, 2.9 mL deionized water, 0.5 mL Folin–Ciocalteu reagent, and 2.0 mL Na₂CO₃ (20%) was left for one hour at ambient conditions (Singleton et al., 1999). During incubation colourless mixture was turned to blue, after which the absorbance was recorded at 765 nm against a reagent blank using UV–Vis spectrophotometer (Dynamica HALO DB-20S, Australia). The readings were compared with the standard gallic acid calibration curve (100–500 mg L^-1) and given as mg kg⁻¹ GAE.

2.5 Quinone content

For the estimation of quinone content, 1 g of pericarp tissue was taken in 4 mL (70%) methanol and left overnight. Then, the extract was subjected to centrifugation at 12000 × g for 20 min and optical density (OD) of supernatant was measured at 437 nm in a UV–vis spectrophotometer. The soluble quinone content was expressed as the absorbance per g (Degl’Innocenti et.al., 2007).

2.6 Total antioxidant capacity

Total antioxidant capacity (TAC) was determined using the CUPRAC assay (Apak et al., 2008). 100 μL of the extracted sample (discussed in total phenol assay) and 1 mL each solution of copper (II) chloride, neocuproine, ammonium acetate buffer and distilled water were mixed and vortexed in a test tube. After 1 h, the absorption was recorded at 450 nm in a UV–vis spectrophotometer, against a blank reagent. The results were expressed as mmol kg⁻¹ Trolox.

2.7 Superoxide (O₂^–) production rate

To determine O₂^– production rate, 1 g pericarp tissue and 4 mL of potassium phosphate buffer (65 mM; pH 7.8) was homogenised and centrifuged at 5000 × g for 10 min. 1 mL of supernatant was then mixed with 0.9 mL of potassium phosphate buffer (65 mM; pH 7.8), 0.1 mL hydroxylammonium chloride (10 mM) and the mixture was incubated for 20 min at 25 °C. Afterwards, sulphanilic acid (17 mM) and α-naphthylamine (7 mM) were added and OD was read at 530 nm. The O₂^– production rate was calculated through a NaNO₂ standard curve and expressed as mmol kg⁻¹ min⁻¹ (Zhao et al., 2008).

2.8 Hydrogen peroxide (H₂O₂) production rate

To determine H₂O₂ production rate, 1 g of pericarp tissue was taken in 10 mL trichloroacetic acid (0.1%; w/v) and centrifuged at 12000 × g for 15 min. 1 mL of the supernatant was then added to 1 mL of potassium phosphate buffer (10 mM; pH 7.0) and 2 mL of potassium iodide (1 M). The OD of the mixture was observed at 390 nm. Readings were compared with the freshly prepared H₂O₂ standard curve to obtain the H₂O₂ concentration (Velikova et al., 2000).

2.9 Membrane leakage

For the estimation of membrane permeability, pericarp pieces were incubated in distilled water for 30 min at 25 °C and the initial conductivity of the solution was recorded using a digital conductivity meter. The same solution was then boiled for 15 min and after cooling another reading of conductivity was taken. The membrane leakage was expressed as a percentage (Ali et al., 2016).

2.10 Malondialdehyde (MDA) content

To assay the malondialdehyde (MDA) content, litchi pericarp (1 g) was homogenized in 15 mL of trichloroacetic acid (10%) and then centrifuged at 10,000 × g for 20 min. Afterwards, 2 mL of each supernatant and 2-thiobarbituric acid (0.6%) was mixed, of which OD was observed at 600, 532, and 450 nm. The MDA contents were calculated and presented as µmol kg⁻¹ (Li, 2000).

2.11 Extraction of crude enzyme and determination of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activities

To determine the activity of polyphenol oxidase (PPO) and peroxidase (POD), 1 g of pericarp tissues was mixed and homogenized with 20 mL of potassium phosphate buffer (50 mM; pH 7.0). The potassium phosphate buffer contained 1% (w/v) polyvinylpyrrolidone (insoluble). The extract was centrifuged at 17,000 × g for 20 min at 4 °C. The supernatant was used as the crude enzyme (Chongchatuporn et al., 2013). For phenylalanine ammonia lyase (PAL) assay, 1 g of pericarp tissues was mixed and homogenized with 20 mL of sodium borate buffer (100 mM; pH 8.0). The sodium borate buffer contained 10% (w/v) polyvinyl poly-pyrrolidone (PVPP), 1 mM EDTA, and 50 mM β-mercaptoethanol. The extract was centrifuged at 17,000 × g for 20 min at 4 °C and the supernatant was collected for PAL assay.

To determine the phenylalanine ammonia lyase (PAL) activity, 0.1 mL of enzyme extract was added in 2.9 mL of sodium borate buffer (0.1 M; pH 8.0) containing L-phenylalanine (3 mM). The mixture was then incubated at 37 °C for 60 min. The increasing absorbance due to the formation of trans-cinnamate was recorded at 290 nm in a UV–vis spectrophotometer. One unit of enzyme activity was defined as the amount that caused an increase of 0.01 in the absorbance per hour. The enzyme activity was expressed as U g⁻¹ protein (Duan et al., 2011).

Catechol (20 mM), prepared in sodium phosphate buffer (0.01 M; pH 6.8), was used as a substrate in PPO assay, while the peroxidase (POD) activity was recorded using guaiacol (4%) as substrate. The methods followed for the determination of PPO and POD were given elsewhere (Jiang et al., 2004; Jiang and Fu, 1999). One unit of enzyme activity is defined as the amount of enzyme causing a 0.1 (PPO) and 0.01 (POD) change in absorbance per min under standard assay conditions. The enzyme activity was expressed as U g⁻¹ protein for both enzymes.

2.12 Statistical analysis

Factorial Completely Randomized Design (FCRD) was used to conduct the experiment. There were three treatments and each treatment was replicated thrice. The statistical significance (p ≤ 0.05) of the difference between means was obtained using Duncan’s Multiple Range Test (DMRT).

3.1 Pericarp browning

The pericarp browning (PB) was 37.5 and 27.5% lower in the litchi fruit treated with 2 and 1 mM MeJA, respectively when compared to control on the final day of storage (Table 1). Reduction in PB primarily occurs with the maintenance of high antioxidant activity and reduced successive disintegration in the pericarp membrane (Jiang et al., 2004). Upon the harvest, the fruit pericarp is gradually desiccated and form micro-cracks on it. The membrane disintegration also occurs with the accumulation of phospholipase-D and ROS (Deshi et al., 2020). Similarly, the lipid peroxidation leads to de-compartmentation resulting in the interaction of oxidative enzymes (PPO and POD present in the cytosol) and substrates (epicatechin and anthocyanin in vacuole). All these changes thereby initiate enzymatic browning reactions and retard red colour in the pericarp (Yang et al., 2011; Barman et al., 2014). Therefore, lower PB in the litchi fruit treated with MeJA could be attributed to reduced moisture losses and enhanced total phenolic and anthocyanin levels in the pericarp (Table 2 and 3).

3.2 Decay incidence

Litchi is vulnerable to many fungal and bacterial diseases during storage, which commonly occurs through cracks or injury in the pericarp (Jiang et al., 2003). An early decay incidence was observed in the control fruit and it increased until the last day of storage (Table 1). The fruit treated with MeJA showed lower decay incidence and on the final day of storage, fruit treated with 2 and 1 mM MeJA, exhibited 76.3 and 65.78% lower decay incidence, respectively, than control fruit (Table 1). PB in litchi fruit has a strong correlation with decay incidence during storage (Deshi et al., 2020). The reduced incidence of decay in the fruit treated with MeJA could be related to the reduced levels of PB as well as enhanced levels of antioxidants and phenolics (Asghari and Aghdam, 2010).

3.3 wt loss

Water loss, due to postharvest physiological activities such as respiration and transpiration, is the primary reason for the loss in fruit weight. These activities are sensitive to changes in relative humidity and prevailing temperature in the storage environment. The browning reactions in the fruit tissue begin with moisture loss and thereby decrease in the fruit weight (Jiang and Fu, 1999). On the final day of refrigerated storage, the litchi fruit treated with MeJA exhibited reduced weight loss by 28.75 and 26.14% in 2 and 1 mM MeJA treated to fruit, respectively, compared to control fruit (Table 2). The lowest weight loss was recorded in fruit treated with 2 mM MeJA, throughout the storage period. Application of MeJA was reported to maintain relatively higher cellular integrity, which further leads to reduced dehydration during storage (Rehman et al., 2018; Habibi et al., 2019). García-Pastor et al. (2020) also reported that the application of MeJA retards the respiration rate in fruit during cold storage and hence delays weight loss.

3.4 Total soluble solids (TSS) and titratable acidity (TA)

Levels of TSS and titratable acidity (TA) are essential factors, which determine the taste and consumer acceptability of litchi fruit. In this study, the litchi fruit treated with MeJA kept under refrigerated storage condition showed inconsistent TSS levels, while titratable acidity declined continuously with increasing storage period. However, when compared to control, the fruit treated with MeJA showed a relatively lower decline in the TA levels (Table 2). On the last day of storage, fruit treated with 2 and 1 mM MeJA retained 66.0 and 58.33% higher titratable acid content, respectively, compared to control fruit. The decline in TA levels is generally associated with fruit senescence processes. Postharvest application of MeJA was reported to retard ripening associated changes and respiration rates in the fruit (García-Pastor et al., 2020).

3.5 Ascorbic acid (AA)

Similarly, the ascorbic acid (AA) content gradually declined with progress in the storage period, irrespective of treatments. Whilst, on the last day of storage, fruit infiltrated with 2 and 1 mM MeJA exhibited 24.0 and 15.44% higher AA content than control ones, respectively (Table 2). Liu et al. (2018) also observed that postharvest MeJA application improves ascorbic acid content in cherry tomato during storage at ambient conditions. The improved AA is regarded as the main bioactive compound in non-enzymatic antioxidant reactions and scavenges reactive oxygen species (ROS), accumulated during stress and different physiological activities (Deshi et al., 2020). The senescence process involves several oxidative reactions and the levels of bioactive compounds such as AA are significantly reduced with progress in senescence. The postharvest application of MeJA retarded such fruit ripening associated changes (García-Pastor et al., 2020).

3.6 Total phenolics

Phenols are another major class of bioactive compounds found abundantly in litchi fruit (Jiang et al., 2003). Even though phenols have many health benefits and exhibit strong natural antioxidant property, they are predominant substrates for oxidative reactions, which ultimately hold responsible for the browning of red pericarp tissue of litchi fruit (Deshi et al., 2020). The total phenolic content (TPC) in litchi pericarp was declined gradually with advancing storage duration, irrespective of the applied treatments (Table 3). However, MeJA treated fruit showed significantly (p < 0.05) higher TPC than control fruit throughout the storage period. On the final day of storage, 2 and 1 mM MeJA treated fruit retained 37.19 and 20.63% higher phenolics as compared to control fruit (Table 3). The decrease in TPC with the extension of the storage period is due to the oxidative reactions associated with fruit senescence processes (Duan et al., 2011). The retention of higher TPC levels in the MeJA treated fruit might be attributed to delayed senescence-associated changes and suppressed PPO and POD activities in the fruit (Barman et al., 2014).

3.7 Anthocyanin and quinone contents

The anthocyanin content in the pericarp was gradually declined, while quinone content was increased, with the extension of the storage duration, irrespective of the treatments (Table 3). At the end of the storage period, fruit treated with 2 mM MeJA exhibited the highest levels of total anthocyanins (2.58 mg kg⁻¹). Fruit that underwent MeJA infiltration retained comparatively higher anthocyanin content than the control fruit. Quinones (ortho-quinones) are the oxidative by-products of phenols and anthocyanins. Yang et al. (2011) reported that MeJA dipping treatment in combination with Sportak fungicide delayed the loss of anthocyanin and (-)-epicatechin during 6 d storage at 25 °C. However, the effect of vacuum infiltrated MeJA alone on anthocyanin content is not reported yet. Accumulation of quinones is one of the prime causes of PB in harvested litchi fruit. The delayed degradation of anthocyanin content and lower levels of quinone accumulation in the litchi fruit treated with MeJA could be correlated with higher total phenol content and retarded activity of oxidative enzymes (Zhang et al., 2018). The levels of quinones accumulated in the MeJA treated fruit was found to be lower, unlike the levels of total anthocyanin content and total phenols, which were higher in MeJA treated fruit. However, there is no clear evidence available on the effect of vacuum infiltrated MeJA on the regulation of quinone contents, which warrants the mode of action that should be investigated.

3.8 Total antioxidant capacity

The antioxidant system in the plants regulates ROS production by continuously scavenging them through antioxidant enzyme activities like SOD, CAT peroxidases as well low molecular non-enzymatic antioxidant compounds such as phenolics, ascorbate and flavonoids (Duan et al., 2011). In this study, there was a gradual decline in total antioxidant capacity (TAC) with increasing storage period, irrespective of the treatments (Table 3). However, MeJA treated fruit maintained significantly (p < 0.05) higher TAC, when compared to control fruit. The fruit treated with 2 and 1 mM MeJA maintained 15.67 and 12.67% higher antioxidant activity, respectively compared to control. Rehman et al. (2018) have opined that MeJA improves the functional quality of harvested fruit by enhancing antioxidant potential. Similarly, Cao et al. (2009) while observing the effect of MeJA on loquat fruit, reported higher levels of bioactive compounds such as phenol MeJA treated fruit and in turn higher antioxidant potential. Therefore, higher TAC values in the litchi fruit treated with MeJA could be associated with the enhanced levels of antioxidant compounds like AA, TPC and total anthocyanins.

3.9 Superoxide anion (O₂^–) and hydrogen peroxide rate (H₂O₂)

Superoxide anion (O₂^–) and hydrogen peroxide (H₂O₂) is the most prevalent reactive oxygen species (ROS) in the plant cell and their production and activity lead to a browning reaction. Irrespective of the treatments applied, ROS concentration increased with an increase in refrigerated storage days (Table 4). However, MeJA-treated fruit exhibited significantly (p < 0.05) lower levels of O₂^– and H₂O₂, throughout the storage period. On the final day of storage, fruit treated with 2 and 1 mM MeJA, exhibited 23.83 and 22.34% lower O₂^– concentration and 32.41 and 21.23% lower H₂O₂ concentration, respectively, when compared to control fruit. The fruit ripening involves the accumulation of ROS due to numerous oxidative reactions as well as catabolic reactions, which give out free radicals as by-products. In postharvest litchi, accumulation of H₂O₂ during storage was also reported by Zhang et al. (2018). They opined that postharvest treatments that delay ROS accumulation could increase the shelf life of litchi. García-Pastor et al. (2020) reported that MeJA application has significantly retarded respiration and ripening associated processes and in turn reduce the accumulation of ROS in the pomegranate fruit. The MeJA also possess the capacity to detoxify ROS by enhancing the activity of antioxidant enzymes under stressful conditions (Mustafa et al., 2016). Phenolics and anthocyanin are considered non-enzymatic antioxidants and have the capability either to scavenge or neutralise free radicals by donating electron (Habibi et al., 2019). In agreement with these findings, MeJA treated fruit exhibited increased levels of AA content, TAC, Anthocyanin, and TPC, which might have actively scavenged ROS in our study.

3.10 Membrane leakage and malondialdehyde (MDA)

Membrane leakage is used to evaluate the permeability and integrity of the cell membrane. During the fruit senescence process, the membrane integrity is degenerated in the litchi pericarp due to the activity of cell wall disintegrating enzymes (Habibi et al., 2019). Whilst MDA is a by-product of membrane disintegration and peroxidation processes (Duan et al., 2011). Irrespective of all treatments, membrane leakage and MDA in litchi pericarp increased with the advancement in storage duration (Table 4). However, fruit subjected to MeJA treatment exhibited a significantly lower leakage rate than control fruit. On the final day of storage, fruit treated with 2 and 1 mM MeJA, exhibited 21.36 and 16.74% lower leakage rate compared to control fruit, respectively. Similarly, on the final day of storage, fruit treated with 2 and 1 mM MeJA showed 7.46 and 6.94% lower MDA content than control fruit, respectively. The results are in agreement with Venkatachalam and Meenune (2015), who reported that MeJA treatment inhibits the activity of cell membrane degrading enzymes in long kong (Lansium parasiticum) fruit under cold storage. The lower levels of membrane leakage and MDA in the fruit treated with MeJA, suggests that the treatment could successfully maintain membrane integrity and limit ROS production in the cells during cold storage (García-Pastor et al., 2020).

3.11 Phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activity

PAL actively involves in the biosynthesis of phenolic compounds in the plant cell. In litchi fruit treated with MeJA, the PAL activity initially increased up to 6 d of storage and then gradually decreased till the end (Table 5). In the case of control fruit, PAL activity decreased continuously with storage duration. On the final day of storage, fruit treated with 2 and 1 mM MeJA, exhibited 56 and 45% higher PAL activity compared to control fruit, respectively. PPO and POD are the key enzymes responsible for senescence-associated oxidative reactions as well as PB in the litchi fruit. PPO and POD levels gradually increased with an increase in the storage duration in the litchi pericarp, irrespective of treatments. However, the MeJA treated fruit exhibited significantly lower levels of the enzymes, when compared to the control fruit (Table 5).

The results also indicated that lower activity of PPO and POD and higher activity of PAL could be attributed to delayed PB in litchi. MeJA application has been reported to delay senescence-associated changes in the fruit thorough decreased activity of oxidizing enzymes during storage (Habibi et al., 2019). The higher levels of total phenols, AA, anthocyanins and total antioxidant capacity could be associated with decreased activity of PPO and POD in the litchi fruit treated with MeJA. The exact mode of action of vacuum infiltrated MeJA on regulating activities of different enzymes in cold-stored litchi fruit is yet to be investigated.