*2.5. Vitamin C, Methionine, L-cysteine, GSH and GSSG*

*2.5. Vitamin C, Methionine, L-cysteine, GSH and GSSG*  The content of two major antioxidants in plants, vitamin C and glutathione (GSH and GSSG) as well as GSH precursors L-cysteine and methionine, presented in Figure 5 and their statistics in Table 1. All the three factors (cultivar, time, and location) have a statistically significant influence on the vitamin C content as well as on the methionine and GSH content. The most interesting statistical difference for our study is the vitamin C content in apple flesh following storage, where cv. 'Majda' flesh contains a higher content of vitamin C in comparison to cv. 'Golden Delicious' (Table S5). In addition, interestingly, there were no statistically significant changes in the vitamin C content in cv. 'Majda' between harvest and storage. The same pattern is also visible with methionine and GSH. With the latter content differed already at harvest (between cultivars within locations). Cysteine was highly influenced by the time and interaction between time and cultivar, its content being statistically lower in cv. 'Golden Delicious' after storage in comparison to harvest at both locations. The GSSG content depended on the cultivar, the content of GSSG in cv. The content of two major antioxidants in plants, vitamin C and glutathione (GSH and GSSG) as well as GSH precursors L-cysteine and methionine, presented in Figure 5 and their statistics in Table 1. All the three factors (cultivar, time, and location) have a statistically significant influence on the vitamin C content as well as on the methionine and GSH content. The most interesting statistical difference for our study is the vitamin C content in apple flesh following storage, where cv. 'Majda' flesh contains a higher content of vitamin C in comparison to cv. 'Golden Delicious' (Table S5). In addition, interestingly, there were no statistically significant changes in the vitamin C content in cv. 'Majda' between harvest and storage. The same pattern is also visible with methionine and GSH. With the latter content differed already at harvest (between cultivars within locations). Cysteine was highly influenced by the time and interaction between time and cultivar, its content being statistically lower in cv. 'Golden Delicious' after storage in comparison to harvest at both locations. The GSSG content depended on the cultivar, the content of GSSG in cv. 'Golden Delicious' flesh representing 69% of the content of GSSG in cv. 'Majda' flesh. Differences are also represented with the heatmap (Figure S2).

'Golden Delicious' flesh representing 69% of the content of GSSG in cv. 'Majda' flesh. Dif-

ferences are also represented with the heatmap (Figure S2).

**Figure 5.** Vitamin C (C vit), methionine (Met), cysteine (Cys), reduced glutathione (GSH), and oxidised glutathione (GSSG) content in apple flesh (mg kg<sup>−</sup>1 FW; mean ± SE) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2) at harvest and following storage. **Figure 5.** Vitamin C (C vit), methionine (Met), cysteine (Cys), reduced glutathione (GSH), and oxidised glutathione (GSSG) content in apple flesh (mg kg−<sup>1</sup> FW; mean <sup>±</sup> SE) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2) at harvest and following storage.

#### *2.6. Phenolic Content 2.6. Phenolic Content*

Phenolic content was determined in flesh (Figure 6), peel (Figure 7), and apple leaves (Figure 8). Determined phenolics were arranged into four groups (for leaves, five) in which they are presented in Figures and mean contents of individual phenols are presented in Table S6 and Figure S3. Hydroxycinnamic acids: Cryptochlorogenic acid, chlorogenic acid, p-coumaric acid, and neochlorogenic acid; dihydrochalcones: Phloridzin, phloretin 2'-O- xylosyl-glucoside, 3-hydroxyphloridzin, and 3-hydroxyphloretin; flavonols: Quercetin-3-rhamnoside, quercetin-3-rutinoside, and quercetin-3-glycoside + quercetin-3-galactoside; and flavan-3-ols: Catechin, epicatechin, procyanidin B1, procyanidin B2 + B4. In the peel of cv. 'Majda', cyanidin-3-galactoside was also determined, but was not included in the results. The 3-hydroxyphloretin was not determined in peel and the phenols neochlorogenic acid, 3-hydroxyphloridzin, 3-hydroxyphloretin, and quercetin-3 rutinoside were not determined in apple flesh. There is a pronounced difference in the phenolic content between cultivars (Table 1). The strongest difference is visible in hydroxycinnamic acid and flavan-3-ol content. The hydroxycinnamic group of phenolics is influenced by all three factors, but there are no interactions between them. Cultivar 'Golden Delicious' has on average 11 times higher hydroxycinnamic acid content in comparison to 'Majda' (Table S8). At L1, the average content of hydroxycinnamic acids represents 75% content of hydroxycinnamic acids at location L2. On the contrary, the flavan-3 ol content was influenced only by factor cultivar, but with high significance. The dihydrochalcone content differed between cultivars only following storage but not at harvest. However, it was also statistically significantly different within both cultivars comparing apple flesh at harvest and following storage (Table S7). Flavonols, on the other hand, were not influenced by time, but by location in addition to the cultivar. At L1, the contrast anal-Phenolic content was determined in flesh (Figure 6), peel (Figure 7), and apple leaves (Figure 8). Determined phenolics were arranged into four groups (for leaves, five) in which they are presented in Figures and mean contents of individual phenols are presented in Table S6 and Figure S3. Hydroxycinnamic acids: Cryptochlorogenic acid, chlorogenic acid, p-coumaric acid, and neochlorogenic acid; dihydrochalcones: Phloridzin, phloretin 2'-Oxylosyl-glucoside, 3-hydroxyphloridzin, and 3-hydroxyphloretin; flavonols: Quercetin-3 rhamnoside, quercetin-3-rutinoside, and quercetin-3-glycoside + quercetin-3-galactoside; and flavan-3-ols: Catechin, epicatechin, procyanidin B1, procyanidin B2 + B4. In the peel of cv. 'Majda', cyanidin-3-galactoside was also determined, but was not included in the results. The 3-hydroxyphloretin was not determined in peel and the phenols neochlorogenic acid, 3-hydroxyphloridzin, 3-hydroxyphloretin, and quercetin-3-rutinoside were not determined in apple flesh. There is a pronounced difference in the phenolic content between cultivars (Table 1). The strongest difference is visible in hydroxycinnamic acid and flavan-3-ol content. The hydroxycinnamic group of phenolics is influenced by all three factors, but there are no interactions between them. Cultivar 'Golden Delicious' has on average 11 times higher hydroxycinnamic acid content in comparison to 'Majda' (Table S8). At L1, the average content of hydroxycinnamic acids represents 75% content of hydroxycinnamic acids at location L2. On the contrary, the flavan-3-ol content was influenced only by factor cultivar, but with high significance. The dihydrochalcone content differed between cultivars only following storage but not at harvest. However, it was also statistically significantly different within both cultivars comparing apple flesh at harvest and following storage (Table S7). Flavonols, on the other hand, were not influenced by time, but by location in addition to the cultivar. At L1, the contrast analysis confirmed the statistical difference between cultivars.

ysis confirmed the statistical difference between cultivars.

interactions (*p* < 0.05).

**Figure 6.** Phenolic content in apple flesh presented in four groups: Hydroxycinnamic acids, dihydrochalcones, flavonols, and flavan-3-ols (mg kg<sup>−</sup>1 FW; mean ± SE) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2) at harvest and following storage. **Figure 6.** Phenolic content in apple flesh presented in four groups: Hydroxycinnamic acids, dihydrochalcones, flavonols, and flavan-3-ols (mg kg−<sup>1</sup> FW; mean <sup>±</sup> SE) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2) at harvest and following storage.

The pattern is quite similar in apple peel, but the differences are not that extreme (Figure 7 and Table 2). Here, the highest difference is in the content of hydroxycinnamic acids. Compared to cv. 'Golden Delicious', cv. 'Majda' has about 13 times lower content of hydroxycinnamic acids (Table S8). Location 1 represents 67% content of hydroxycinnamic acids at L2. There were no statistical differences between cultivars in dihydrochalcone and flavonol content, but the latter did vary between locations. Again, flavan-3-ol content differed in cv. 'Golden Delicious' (four times higher content) in compar-The pattern is quite similar in apple peel, but the differences are not that extreme (Figure 7 and Table 2). Here, the highest difference is in the content of hydroxycinnamic acids. Compared to cv. 'Golden Delicious', cv. 'Majda' has about 13 times lower content of hydroxycinnamic acids (Table S8). Location 1 represents 67% content of hydroxycinnamic acids at L2. There were no statistical differences between cultivars in dihydrochalcone and flavonol content, but the latter did vary between locations. Again, flavan-3-ol content differed in cv. 'Golden Delicious' (four times higher content) in comparison to cv. 'Majda'.


ison to cv. 'Majda'. **Table 2.** Two-factor ANOVA for cultivar (Cul: 'Golden Delicious' and 'Majda') and location (Loc: L1 and L2) and their **Table 2.** Two-factor ANOVA for cultivar (Cul: 'Golden Delicious' and 'Majda') and location (Loc: L1 and L2) and their interactions (*p* < 0.05).

> Dihydrochalcones \*\*\* \* NS Flavonols \*\*\* . NS ., statistically significant differences at *p* < 0.1; \*, statistically significant differences at *p* < 0.05; \*\*, statistically significant differences at *p* < 0.01; \*\*\*, statistically significant differences at *p* < 0.001; NS: Not significant.

., statistically significant differences at *p* < 0.1; \*, statistically significant differences at *p* < 0.05; \*\*, statistically significant

differences at *p* < 0.01; \*\*\*, statistically significant differences at *p* < 0.001; NS: Not significant.

Flavan-3-ols \*\*\* \*\*\* NS

**Figure 7.** Phenolic content in apple peel presented in four groups: Hydroxycinnamic acids, dihydrochalcones, flavonols, and flavan-3-ols (mg kg<sup>−</sup>1 FW; mean ±SE) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2). **Figure 7.** Phenolic content in apple peel presented in four groups: Hydroxycinnamic acids, dihydrochalcones, flavonols, and flavan-3-ols (mg kg−<sup>1</sup> FW; mean <sup>±</sup>SE) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2).

In apple leaves, the differences are not as drastic as in fruits but are still present (Figure 8 and Table 2). All the phenolics were influenced by both cultivar and location, with hydroxycinnamic acids and flavonols having weak, statistically significant differences regarding location. However, there were no interactions between factors cultivar and location. Cultivar 'Golden Delicious' had twice as much arbutin, 1.4 times higher flavonol content and 2.9 times higher flavan-3-ol content as cv. 'Majda' (Table S8). On the other hand, cv. 'Golden Delicious' had 70% and 92% content of the cv. 'Majda' content of hy-In apple leaves, the differences are not as drastic as in fruits but are still present (Figure 8 and Table 2). All the phenolics were influenced by both cultivar and location, with hydroxycinnamic acids and flavonols having weak, statistically significant differences regarding location. However, there were no interactions between factors cultivar and location. Cultivar 'Golden Delicious' had twice as much arbutin, 1.4 times higher flavonol content and 2.9 times higher flavan-3-ol content as cv. 'Majda' (Table S8). On the other hand, cv. 'Golden Delicious' had 70% and 92% content of the cv. 'Majda' content of hydroxycinnamic acids and dihydrochalcones, respectively.

droxycinnamic acids and dihydrochalcones, respectively.

**Figure 8.** Phenolic content in apple leaves presented as arbutin and four groups: Hydroxycinnamic acids, dihydrochalcones, flavonols, and flavan-3-ols (mg kg<sup>−</sup>1 DW) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2). **Figure 8.** Phenolic content in apple leaves presented as arbutin and four groups: Hydroxycinnamic acids, dihydrochalcones, flavonols, and flavan-3-ols (mg kg−<sup>1</sup> DW) for cultivars 'Golden Delicious' and 'Majda' at two different locations (L1 and L2).
