Effect of Postharvest Methyl Jasmonate Treatment on Early-Matured “Hass” Avocado Fruit Exocarp Colour Development during Ripening

Poor exocarp colour development is a common postharvest problem for early harvested “Hass” avocado fruit during ripening, which affects fruit quality and consumer preference. Therefore, measures to improve “Hass” avocado fruit colour developments are of great importance in the industry. This study investigated the effectiveness of postharvest methyl jasmonate treatment to improve early matured “Hass” avocado fruit exocarp colour during ripening. The results showed that T1 (10 µmol∙L −1 ) and T2 (100 µmol∙L −1 ) MeJA treatment increased visual colour, and decreased objective colour parameters (L*, C* and h˚) during ripening when compared with control fruit. Moreover, MeJA treated “Hass” avocado fruits had lower total chlorophyll content and higher total anthocyanin and cyanidin-3-O-glucoside concentration during ripening. In conclusion, “Hass” avocado fruit post-harvest treated with either T1 (10 µmol∙L −1 ) or T2 (100 µmol∙L −1 ) MeJA concentration improved exocarp quality attributes such as colour parameters (L*, C* h˚ and visual colour) and pigments (total anthocyanin and cyanidin-3-O-glucoside) during ripening, therefore, can be recommended for avocado fruit.

and contributes significantly to fruit marketing [1] [2]. However, "Hass" avocado fruit from South Africa has been showing poor exocarp colouration during ripening, therefore, compromising quality fruit and consumer preferences.
Mathaba et al. [2] found that "Hass" avocado fruit harvested at early maturity tended to show poor exocarp colour development during ripening. In general, "Hass" avocado fruit changes exocarp colour from green to purple and eventually black, mainly owing to chlorophyll degradation and synthesis and anthocyanin accumulation [1] [3]. In "Hass" avocado fruit exocarp, cyanidin-3-O-glucoside is the most abundant specific anthocyanin pigment during ripening [1]. According to Ashton et al. [4], cyanidin-3-O-glucoside is an anthocyanin compound responsible for the purple and black colour development during the ripening of "Hass" avocado fruit. Recently, there is an interest in exploring postharvest treatment which triggers the enhancement of anthocyanin synthesis and accumulation, consequently, leading to improved early matured "Hass" fruit exocarp colour development during ripening.
In this context, methyl Jasmonate (MeJA), a member of the cyclopentanone compound, has been established as a signalling molecule with a positive impact on the colour development of various fruits [5]. In the literature, it has been reported that preharvest or postharvest treatment with MeJA can influence colour development by enhancement of anthocyanin biosynthesis and accumulation in numerous crops including apple [5], pepper [6], grape [7], blueberries [8], eggplant [9] and tomato [10].
In table grapes, postharvest MeJA treatment at 1, 0.1 and 0.01 mM accelerated ripening and increased total phenolic and anthocyanin concentration [11]. In Blackberries, Wang, Bowman [12] showed that preharvest MeJA treatment significantly increased anthocyanin, total phenolic content and antiproliferative activity. Furthermore, Muengkaew et al. [13] found that dipping mango fruit in MeJA at 80 µL•mL −1 resulted in higher phenylalanine ammonia-lyase (PAL) enzyme activity, phenolic compound concentration and higher anthocyanin concentration when compared control fruit. Therefore, the objective of this study was to investigate the effectiveness of postharvest MeJA treatment applied at different concentrations for enhancing anthocyanin concentration, subsequently, to improve purple colour development of early matured "Hass" avocado fruit during ripening.

Plant Materials
In 2018, avocado fruit "Hass" were harvested from Nico Swart Farm in Kiepersol

Postharvest MeJA Treatment
The experiment was conducted as a completely randomized design (CRD) with MeJA solution. The treated fruits were allowed to dry at room temperature (25˚C) for 1 hour. Thereafter, treated and control fruits were further packed in plastic crates and stored at 5.5˚C with relative humidity (RH) of 98% for 28 days.
After 28 days, fruits were removed from cold storage and transferred to room temperature at 25˚C and allowed to ripen. Five fruits from each treatment were randomly sampled at every evaluation day (0, 2, 4 and 6 at 25˚C), subsequently, exocarp tissues of the samples were frozen in liquid nitrogen and cold-stored at −21˚C until further analysis.

Total Chlorophyll Analysis
Total chlorophyll was extracted according to Lichtenthaler [15]. Briefly, 0.5 g powder exocarp tissues were extracted with 10 ml of 80% acetone, kept on ice for 30 minutes then centrifuged at 6000× g for 5 minutes. Total chlorophyll contents were estimated by reading the absorbance at 470, 646 and 663 nm in a UV-visible spectrophotometer (Jenway, UK). The total chlorophyll contents were calculated by adding C a and C b using equations as follows: where C a and C b represents chlorophyll a and b.

Total Anthocyanin and Cyanidin-3-O-Glucoside Analysis
Extraction was done according to Cox, McGhie [1], avocado exocarp tissues were milled to powder under liquid nitrogen and 0.5 g was extracted with 5 ml of 10% acetic acid/methanol (v/v) at room temperature. Therefore, centrifugation at 3000× g for 10 minutes, the supernatant was diluted 1:1 with methanol:water:acetic acid (50:50:10, v/v/v). The pH differential method previously described by Giusti and Wrolstad [16] was used to determine total anthocyanin content. The diluted 1:1 supernatant was filtered through 0.45 µm nylon filters into clean vials and diluted with 1 µl of potassium chloride buffer (pH 1.0 ) and sodium acetate buffer (pH 4.5 ). The mixtures were allowed to settle in the dark for 10 minutes, subsequently, absorbance values of each buffer mixture were measured at 530 and 700 nm in a UV-visible spectrophotometer (Jenway, UK). The total anthocyanin was calculated using the equation.

Statistical Analysis
The analysis of variance (ANOVA) was performed with Genstat 16th version (VSN International, UK). The Least Significant Difference (LSD) was used to calculate the p-value at 95% confidence interval around the difference between treatments. Furthermore, Pearson correlation analysis was performed to determine the relationship between firmness, colour parameters, and exocarp pigments.

Colour (Subjective and Objective Parameters)
As "Hass" avocado fruit ripens, it is supposed to acquire a purple exocarp colour, which later develops to black colour while the green colour is degraded. In this study, there was a significant difference (p = 0.0228) in subjective (visual colour) between studied treatments during ripening. In general, there was an increase in exocarp visual colour for T1 (10 µmol•L −1 ) and T2 (100 µmol•L −1 ) MeJA treatment when compared with control fruit (Figure 1(a)). In this study, MeJA treated fruit showed higher visual colour (5-purple), while control showed lower visual colour (4-violet) on day 6 at 25˚C. In terms of objective exocarp colour change, MeJA treated and control fruit showed no significant difference for L* (p = 0.3997), C* (p = 0.0665) and h˚ (p = 0.0647) during ripening ( Figures  1(b)-(d)). However, the objective exocarp colour parameters (L*, C* and h˚) decreased for all the treatments during ripening. Moreover, control fruits showed higher L* values than MeJA treated fruit on day 6 at 25˚C, which corresponded with green colour maintenance.
The C* values did not differ between MeJA treated and control fruit on day 6 at 25˚C. However, the h˚ values for control were higher when compared with MeJA treated fruit on day 6 at 25˚C. In addition, the correlations obtained between visual colour and objective colour parameters were negative and significant (p < 0.001), which could mean that exocarp colour change was dependent on decreased objective colour parameters (L*, C* and h˚) ( Table 1). Our results were in agreement with previous researchers on "Hass" avocado fruit [1] [2] [4] who found that objective exocarp colour parameters (L*, C* and h˚) decrease to their lowest values during ripening. Furthermore, objective exocarp colour parameters (L*, C* and h˚) of MeJA treated fruit were lower than those of control fruit on days 4 and 6 at 25˚C. Such results indicated that MeJA treatment stimulated exocarp purple colour development for "Hass" avocado fruit during ripening. These results could be attributed to the role of MeJA in regulating anthocyanin biosynthesis and implied that an improved exocarp colour development was enhanced by MeJA treatment when compared with control fruit. Methyl jasmonate promotes anthocyanin biosynthesis by up-regulating the transcription of related genes [11]. This could be the reason for the total anthocyanin to be highly and positively correlated (p < 0.001) with cyanidin-3-O-glucoside for MeJA treated fruit but not for control fruit.

Total Chlorophyll Content
In this study, the total exocarp chlorophyll content in all fruit decreased continuously during ripening, however, different contents were observed among the three treatments ( Figure 2). Our findings showed that T1 (10 µmol•L −1 ) MeJA concentration markedly accelerated this trend, with a significant difference (p < 0.0001) between MeJA treated and control fruit. After 6 days at 25˚C, mean chlorophyll degradation for control fruit was 2.67 mg 100 g −1 DW, while in T1 (10 µmol•L −1 ) and T2 (100 µmol•L −1 ) MeJA treated were 2.38 and 3.36 mg 100 g −1 DW, respectively. However, fruit treated with T2 (100 µmol•L −1 ) MeJA concentration exhibited higher total chlorophyll contents, while lower contents were found in control and T1 (10 µmol•L −1 ) MeJA treated fruit after 4 and 6 days at 25˚C, respectively. Chlorophyll degradation for "Hass" avocado fruit often relates to colour development during ripening [1]. As the fruit ripen and soften,  (Table 1). The relationship between MeJA treatment and increased anthocyanin accumulation was also reported in other fruit species [18] [19]. It has been reported that preharvest and postharvest MeJA treatment increased activities of phenylalanine ammonia-lyase (PAL) enzyme which is responsible for polyphenol synthesis including anthocyanin [20]. For instance, treatment of MeJA combined  teinase inhibitors and Chitinase, therefore, triggering the accumulation of anthocyanin in grapevine [21]. In strawberry and blackberry, bioactive compounds including; total phenolic, anthocyanin and carotenoids were enhanced by post-harvest MeJA treatment [19] [22]. In mango fruit, MeJA treatment induced higher PAL activity, in turn, increased phenolic and anthocyanin synthesis and accumulation [13]. In our current research work, MeJA supposedly induced PAL activity, and thereby, cyanidin-3-O-glucoside synthesis and accumulation, consequently, improved early matured "Hass" avocado exocarp colour development during ripening.

Fruit Firmness
Fruit firmness loss of avocado is an important ripeness indicator and directly affects shelf-life. A significant (p = 0.0371) decreasing trend in fruit firmness was found amongst all the treatments. In the present work, firmness loss was increased gradually with days to ripening, regardless of treatment ( Figure 4).
However, postharvest application with MeJA contributed collaboratively to accelerated firmness loss for "Hass" avocado fruit during ripening when compared with control fruit. Similar results have been reported by Cao et al. [23], who observed that firmness level decreased for loquat (Eriobotrya japonica Lindl.) fruit, regardless of treatment during storage. In the present study, fruit treated with lulase, PG and PME and β-galactosidase has also been reported for postharvest application of MeJA in "Beauty" blueberry fruit [26].

Conclusion
This study revealed that MeJA treatment improved the exocarp colour development of "Hass" avocado fruit during ripening. The results demonstrate that post-harvest treatment with MeJA improved exocarp attributes such as colour chromaticity (L*, C* h˚ and visual colour) and pigments (total anthocyanin and cyanidin-3-O-glucoside) during ripening, therefore, can be recommended for avocado fruit.