*2.4. Statistical Analysis*

The experiment used a completely randomized design (CRD) with three replicates; each replicate contained four mango transplants. The statistical analysis was performed using the R software, version 4.0.5, R Core Team, Vienna, Austria [35]. The main treatment effects at each sampling time were analyzed, and the means were compared by Duncan's multiple range tests [36] at a significance level of 0.05. Pearson's correlation coefficient was also calculated to specify associations between any measured parameters at each sampling time in response to chilling and SA application.

#### **3. Results and Discussion**

The effects of the exogenous SA application on chilled "Seddik" mango transplants were observed. Defoliation percentages for all treatments, after 21 days of exposure to chilling stress, were significantly high (*p* ≤ 0.05) compared to those of the negative control (8.33%), but the chilled transplants pretreated with 1.5 mM L−<sup>1</sup> SA yielded an acceptable defoliation percentage (23.33%). The positive control revealed the highest defoliation percentage (45.14%) after 21 days of exposure to chilling stress (Figure 2A). Chilling temperatures (lower than 10 ◦C) cause many physiological changes in chillingsensitive plants, inducing CI and even mortality in tropical and subtropical species [37]. The plants exhibited a steady increase in leaf fall in the early stages of cold stress exposure and, as days progressed, they became even more defoliated [38]. Similarly, the chilling injury index (CII) of all chilled transplants not treated (positive control) or pretreated with SA was significantly higher (*p* ≤ 0.05) than that of the negative control (Figure 2B). Thus, SA pretreatment at 1 and 1.5 mM alleviated mango transplants' CI symptoms (Figure 2E,F, respectively) compared with the positive control (Figure 2D). In fact, the application led to a vital reduction in CI incidence [2,39]. Exposure of the mango transplants to chilling stress for 72 h critically affected plant photosynthetic pigments, chlorophyll a, b, and total pigments, even in transplants pretreated with SA. However, after six days of recovery, the SA-treated mango transplants, specifically, those treated with SA at 1.5 mM, showed pigment values similar to those of the negative control (normal growth conditions). Hence, SA application mitigated the chilled mango transplants' chilling stress during the recovery period (Figure 3A–C). The lowest total chlorophyll values were 8.99 and 8.87 mg g−<sup>1</sup> for positive control treatment after zero and six days of chilling stress, respectively. Similarly, the chlorophyll stability index (CSI) was the lowest for the untreated transplants (positive control) exposed to chilling stress; SA increased mango leaves' CSI compared with the positive control (Figure 3D).

**Figure 2.** Defoliation percentage for each treatment after 21 days of chilling stress (**A**), chilling injury index (**B**), chilling injury (CI) symptoms in mango transplants, negative control (**C**), CI symptoms in chilled mango transplants, positive control (**D**), CI symptoms in chilled mango transplants pretreated with 1 mM SA (**E**) and 1.5 mM SA (**F**). Bars with different letters represent significantly different data at 95% confidence, as determined by Duncan's Multiple Range Test. Error bars represent the standard deviation.

Under low-temperature conditions, chlorophyll-degrading enzymes' (chlorophyllases) activity increases, and their biosynthesis is inhibited, leading to a decrease in chlorophyll content in chilled plants [40,41].

The leaves' low chlorophyll content at low temperatures can be interpreted as a lack of photosynthetic efficiency [42,43]. Moreover, the reduction in photosynthetic capacity at low temperatures is associated with a decrease in PSII quantum efficiency, the primary target of damage at low temperatures [1,44]. Chilling damage occurs when membranes acquire more saturated fatty acids due to the exposure to low temperatures [44–46]. In Figure 4A, it is discernible that the transplants exposed to chilling stress showed an increase in electrolyte leakage percentage after zero (52.99%) or six days (64.51%) compared to those pretreated with SA. The negative control recorded the lowest electrolyte leakage percentage values after zero (28.49%) and six days (28.05%) of exposure to chilling stress.

**Figure 3.** Changes in leaf chlorophyll content; chlorophyll a content (**A**); chlorophyll b content (**B**); total chlorophyll (**C**), and chlorophyll stability index (**D**) in mango transplants under the studied treatments. Different lower-case letters indicate statistical differences between treatments at zero days after 72 h of chilling stress exposure, while the upper-case letters indicate significant differences between treatments at six days at 95% confidence, as determined by Duncan's Multiple Range Test. Error bars represent the standard deviation.

**Figure 4.** Changes in electrolyte leakage (**A**) and membrane stability index (**B**) of mango transplants' leaves under the studied treatments. Different lower-case letters indicate statistical differences between treatments at zero days after 72 h of chilling stress exposure, while the upper-case letters indicate significant differences between treatments at six days at 95% confidence, as determined by Duncan's Multiple Range Test. Error bars represent the standard deviation.

Contrarily, the membrane stability index was the highest for the negative control compared to the positive control, with the lowest values (47.01 and 35.49%) after zero and six days of chilling stress exposure, respectively (Figure 4B). Usually, electrolytes leakage is used to assess the chilling damage [47]. Guinn [48] suggested that the increase in electrolyte leakage is likely due to chilling-induced water stress. Furthermore, increased electrolyte leakage from chilled plants was attributed to membrane deterioration and corresponded to the presence of leaked inorganic and organic ions [46,49–52]. SA role in maintaining the fatty acids' content and ratio in the cell membranes could explain its protection of the cell membrane structure [53]. Differences in total sugar content were not statistically significant but accrued gradually with increased SA concentrations, specifically, after six days of recovery, in the chilled mango transplants compared with the positive control (Figure 5A). Generally, plants amass many relevant solutes, such as soluble sugars and amino acids, in response to cold and other osmotic stresses [54–56]. During the recovery period, the highest significant (*p* ≤ 0.05) total phenolic content was recorded in the positive control, with 3.76 mg g<sup>−</sup>1, and the lowest (*<sup>p</sup>* ≤ 0.05) in the negative control, with 1.81 mg g<sup>−</sup>1. The same trend was also evident after exposure to chilling stress for all treatments (Figure 5B). The transplants pretreated with increased SA concentrations before being subjected to chilling had a lower proline content after zero or six days of stress exposure, as shown in Figure 5C. Rivero et al. [57] observed that mango tissues accrued phenolic compounds under cold stress. A decreased amount of phenolics was observed in SA-pretreated transplants, depicting the effect of SA in alleviating CI in mango under chilling. This finding agrees with Han et al. [58]. However, Wongsheree et al. [45] found that total phenolic compounds in lemon basil leaves, whether young or mature, were not affected by chilling stress exposure at 4 ◦C. Under cold stress, exogenous SA application caused an increase of soluble carbohydrates in *Phaselous vulgaris* [59]. SA treatment substantially increased solutes and total soluble sugars, and these osmolytes promoted cryostability in the cell membranes, protecting the plants from cold stress [60,61]. Moreover, the stress conditions increased proline metabolism, attributable to an increase in proline biosynthesis enzymes (pyrolline-5-carboxylate reductase and -glutamyl kinase) [62,63]. Sayyari et al. [64] reported that SA ameliorated CI by inhibiting proline accumulation; the variation in proline content in response to chilling stress primarily depended on the plant genotype [64,65].

Concerning DPPH radical-scavenging activity, SA application positively impacted the chilled mango transplants during recovery by enhancing the function of DPPH. It increased with increases in SA concentration, and the lowest value was measured for the positive control (Figure 5D). The induction of DPPH scavenging activity in chilled mango transplants by SA depended on the concentration applied. This was also observed for banana [66], mango [67,68], and lemons [69] when cold-exposed fruits were treated with SA. Under chilling conditions, SA pre-treatment reduced SOD activity in mango leaves (Figure 6A). After 72 h of exposure to chilling stress, SOD activity significantly (*p* ≤ 0.05) decreased compared to that in the negative control (0.81 U g<sup>−</sup>1) but significantly (*<sup>p</sup>* ≤ 0.05) increased with the gradual increase of SA concentration (0.65, 0.93, and 1.00 U g−<sup>1</sup> with 0.5, 1, and 1.5 mM SA, respectively). The positive control exhibited the lowest significant (*<sup>p</sup>* ≤ 0.05) value (0.56 U g−1). This trend was also evident for the recovery period with fewer responses, whereas the negative control showed the highest (0.80 U g−1) and the lowest values (0.38 U g<sup>−</sup>1). The SOD values in chilled transplants pretreated with SA were 0.42, 0.61, and 0.64 U g−<sup>1</sup> with 0.5, 1, and 1.5 mM SA, respectively. Similarly, chilling stress (positive control) significantly (*p* ≤ 0.05) reduced CAT activity during recovery compared to the negative control, but SA pre-treatment gradually increased CAT activity (*p* ≤ 0.05) in chilled mango transplants (Figure 6B). Under chilling conditions, POX activity in mango leaves was significantly (*p* ≤ 0.05) high for the positive control, reaching the highest value (0.73 U g−1) compared to the negative control (0.104 U g−1). As expected, SA pre-treatment gradually decreased POX activity after six days of chilling stress to its normal level (0.104 U g<sup>−</sup>1) with 1.5 mM L−<sup>1</sup> SA treatment (Figure 6C).

**Figure 5.** Changes in total sugar (**A**), total phenolic content (**B**), proline content (**C**), and DPPH (**D**) in mango transplant leaves. Different lower-case letters indicate statistical differences between treatments at zero days after 72 h of chilling stress exposure, while the upper-case letters indicate significant differences between treatments at six days at 95% confidence, as determined by Duncan's Multiple Range Test. Error bars represent the standard deviation.

**Figure 6.** Changes in the activities of superoxide dismutase (**A**), catalase (**B**), peroxidase (**C**), and polyphenol oxidase (**D**) in mango transplant leave. Different lower-case letters indicate statistical differences between treatments at zero days after 72 h of chilling stress exposure, while the various upper-case letters indicate significant differences between treatments at six days at 95% confidence, as determined by Duncan's Multiple Range Test. Error bars represent the standard deviation.

Moreover, PPO activity in chilled mango leaves was also significantly (*p* ≤ 0.05) high after exposure to chilling. However, SA gradually decreased the enzyme activity by about half compared to the negative control. After six days of exposure to chilling stress, PPO activity in chilled mango leaves was significantly (*p* ≤ 0.05) higher, and the SA treatments significantly (*p* ≤ 0.05) reduced PPO activity until it approximately returned to the normal level compared to the negative control (Figure 6D).

The untreated mango leaves exhibited lower SOD and CAT enzyme activities after six days of chilling stress exposure than the treated ones. It implies less protection against membrane oxidation in untreated leaves [45]. Accordingly, a positive correlation between SOD and CAT enzyme activities was detected after zero (*r* = 0.34) and six days (*r* = 0.81) of chilling stress exposure (Figure 7A,B). Generally, SA treatment effectively alleviated the chilled mango transplants' CI compared to the negative control, reaching the normal levels after six days of stress (recovery). These findings are aligned with those of Chen et al. [9] and Khademi et al. [66], who found that SA treatment effectively reduced banana CI by maintaining membrane integrity and improving antioxidants' activity. SOD and CAT enzymatic activities exhibited the same trend. SOD, the primary line of defense against ROS-induced oxidative damages, catalyzes the dismutation of two superoxide radicals into H2O2 and O2. CAT converts H2O2 into H2O and O2. Still, POX enables H2O2 oxidation and yields water and another oxidizing molecule. This was evident in the highly positive significant correlation (*p* < 0.001) between POX enzyme activity and EL (*r* = 0.77) after 72 h of exposure to chilling stress (Figure 7A). PPO causes tissue browning in most horticultural crops by oxidizing phenolic compounds to quinones. CI alleviation by SA application decreased PPO activity and increased antioxidant systems and specific bioactive chemicals' concentration, as reported for banana [70], cherry [71], litchi [72], pomegranate [18], and wax apple [73]. After six days of exposure to chilling, PPO had significant (*p* < 0.001) correlations with EL, total phenol, and proline (Figure 7B). Meanwhile, it showed strong negative correlations with MSI, plant photosynthetic pigments (chlorophyll a, b, and total), SOD, and CAT. SA treatment of cold-stressed plants altered the activities of different enzymatic antioxidants such as CAT, SOD, and POX [74,75]. In the correlation analysis, a significant positive correlation (*p* < 0.001) between CAT, chlorophyll a, b, MSI, SOD, and total sugar (Figure 7B) was found. SA was shown to enable some crops to recover from cold damage by regulating antioxidative mechanisms [39,59,76,77].

**Figure 7.** Pearson's correlation coefficient analysis for each sampling time, (**A**) for day zero and (**B**) for day six after 72 h of chilling stress exposure. R is presented in different colors; the legend shows the color range of different R values with \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 as indicators of statistical significance.
