*2.7. PPO and POX*

*2.7. PPO and POX*  The PPO and POX activities are presented in Figure 9 and statistics in Table 1. There were statistically significant differences in the PPO activity in all three factors. However, the contrast analysis showed no statistically significant differences between cultivars (Table S9). There was a difference in the enzyme activity between apple flesh at harvest and following storage. With POX, time was the only statistically different factor and culti-The PPO and POX activities are presented in Figure 9 and statistics in Table 1. There were statistically significant differences in the PPO activity in all three factors. However, the contrast analysis showed no statistically significant differences between cultivars (Table S9). There was a difference in the enzyme activity between apple flesh at harvest and following storage. With POX, time was the only statistically different factor and cultivar:location was the statistically significant interaction.

var:location was the statistically significant interaction.

**Figure 9.** Activity of PPO and POX (ΔA min<sup>−</sup>1) in apple flesh of cultivars 'Golden Delicious' and 'Majda' at harvest and following storage. **Figure 9.** Activity of PPO and POX (∆A min−<sup>1</sup> ) in apple flesh of cultivars 'Golden Delicious' and 'Majda' at harvest and following storage.

#### **3. Discussion**

**3. Discussion**  The results show some promising differences between cultivars. Cultivar 'Majda' contains a lower sugar content than cv. 'Golden Delicious'. Cultivars also differ in individual sugar composition. Even though according to Aprea et al. [22] sorbitol correlates to sweetness the most, its low share in total sugar composition and lower fructose and glucose content in cv. 'Majda' do not contribute so much to the sweetness. In addition, the study by Rymenants et al. [23] reported that the perceived sweetness was greatly influenced by the acidity and vice versa. Apples with low acidity are perceived to be sweeter, whereas apples with a strong acidic taste are perceived as less sweet. This is most likely behind the cv. 'Majda' taste perception, further supported by Aprea et al. [22], who assessed a negative correlation between malic acid and perceived sweetness (r = −0.449). The higher malic acid content in cv. 'Majda' could also be connected to non-browning. In the study by Morimoto et al. [24], they reported on the connection between high acidity alleles and the alleles for non-browning. The major locus controlling apple fruit acidity has been designated as Ma (malic acid), where Ma corresponds to the dominant high acidity or the low pH allele and ma is the low acidity or the high pH allele [25]. For cv. 'Majda', it was also believed that the high acidity and low pH are the main factors for its lack of browning. However, in accordance with the previously mentioned data, it might just be the conse-The results show some promising differences between cultivars. Cultivar 'Majda' contains a lower sugar content than cv. 'Golden Delicious'. Cultivars also differ in individual sugar composition. Even though according to Aprea et al. [22] sorbitol correlates to sweetness the most, its low share in total sugar composition and lower fructose and glucose content in cv. 'Majda' do not contribute so much to the sweetness. In addition, the study by Rymenants et al. [23] reported that the perceived sweetness was greatly influenced by the acidity and vice versa. Apples with low acidity are perceived to be sweeter, whereas apples with a strong acidic taste are perceived as less sweet. This is most likely behind the cv. 'Majda' taste perception, further supported by Aprea et al. [22], who assessed a negative correlation between malic acid and perceived sweetness (r = −0.449). The higher malic acid content in cv. 'Majda' could also be connected to non-browning. In the study by Morimoto et al. [24], they reported on the connection between high acidity alleles and the alleles for non-browning. The major locus controlling apple fruit acidity has been designated as Ma (malic acid), where Ma corresponds to the dominant high acidity or the low pH allele and ma is the low acidity or the high pH allele [25]. For cv. 'Majda', it was also believed that the high acidity and low pH are the main factors for its lack of browning. However, in accordance with the previously mentioned data, it might just be the consequence of heritability rather than the key reason for non-browning.

quence of heritability rather than the key reason for non-browning. The difference in color change between cultivars is evident. Similar results were also reported by Joshi et al. [26] for cultivar 'Eden'. In our research, the halves and pomace of cv. 'Majda' both had minimal change in color in comparison to cv. 'Golden Delicious'. The difference in color change between cultivars is evident. Similar results were also reported by Joshi et al. [26] for cultivar 'Eden'. In our research, the halves and pomace of cv. 'Majda' both had minimal change in color in comparison to cv. 'Golden Delicious'. Next, we will discuss the main possible causes for this trait.

Next, we will discuss the main possible causes for this trait. Both vitamin C and GSH are major antioxidants and this characteristic is also recognized and used in the food industry [2]. However, according to Nicolas [27] the endogenous vitamin C has a marginal role in the prevention of browning. On the contrary, Joshi et al. [26] did find the correlation between the vitamin C content and the whiteness index. In our study, the cultivars did not differ in vitamin C content at harvest, but they did differ in retaining of vitamin C following storage. Following storage, the content of vitamin C remained higher in the cv. 'Majda' fruit. Davey and Keulemans [19] reported that cultivars differ substantially in their ability to maintain vitamin C during storage. In their study, the 'Golden Delicious' vitamin C decreased following storage, whereas in a few other cultivars it increased. Furthermore, they report on the correlation between fruit vitamin C contents and the harvest date, such that cultivars with the highest vitamin C contents were harvested latest in the season and the lowest contents were found among the early culti-Both vitamin C and GSH are major antioxidants and this characteristic is also recognized and used in the food industry [2]. However, according to Nicolas [27] the endogenous vitamin C has a marginal role in the prevention of browning. On the contrary, Joshi et al. [26] did find the correlation between the vitamin C content and the whiteness index. In our study, the cultivars did not differ in vitamin C content at harvest, but they did differ in retaining of vitamin C following storage. Following storage, the content of vitamin C remained higher in the cv. 'Majda' fruit. Davey and Keulemans [19] reported that cultivars differ substantially in their ability to maintain vitamin C during storage. In their study, the 'Golden Delicious' vitamin C decreased following storage, whereas in a few other cultivars it increased. Furthermore, they report on the correlation between fruit vitamin C contents and the harvest date, such that cultivars with the highest vitamin C contents were harvested latest in the season and the lowest contents were found among the early cultivars. It is suggested that this is linked to the cultivar's capacity for longer storage. However, at this point, we must emphasize on the better storage ability of cultivar 'Golden

Delicious' in comparison to that of cultivar 'Majda' in CA storage (non-published data and experience from growers). The contrast analysis also showed the difference in vitamin C content in the flesh of cv. 'Golden Delicious' between locations, whereas the content of vitamin C of cv. 'Majda' apples did not vary between locations. It is known that the content of vitamin C is highly influenced by temperature and radiation [28], so this might add an explanation to the higher vitamin C content at L2, but then again, since cv. 'Majda' did not show the similar response, this is just a speculation that needs to be addressed in future studies. What we can see, though, is also a higher content of the reduced form of GSH in cv. 'Golden Delicious' at harvest at L2. Therefore, location did influence these two antioxidants in cv. 'Golden Delicious'. However, what stands out with GSH is the statistically significant higher content of GSH in cv. 'Majda' at both locations and at both times. What is more, if we look at the phenolic content in flesh, we can significantly see a lower content of hydroxycinnamic acids. In a review on the role of glutathione in winemaking, Kritzinger et al. [29] reported on an interesting observation, the ratio of hydroxycinnamic acid and GSH (HCA/GSH) represents a good indication of the grape must susceptibility to oxidation. A ratio of 0.9–2.2 characterizes a lightly colored must, while the medium and dark must are characterized by 1.1–3.6 and 3.8–5.9 HCA/GSH ratio, respectively. If we compare the HCA/GSH ratio in our samples we can see a great difference, 2.7–3.2 and 0.2–0.3, for cv. 'Golden Delicious' and cv. 'Majda', respectively. Therefore, we have two aspects we need to take into account when discussing the lack of oxidation in cv. 'Majda': Low hydroxycinnamic acid content and high GSH content.

In addition to the low hydroxycinnamic acid, the flavan-3-ols content shows the significant differences in the content of main phenols responsible for browning, namely chlorogenic acid, catechin, and epicatechin [30,31]. Furthermore, at harvest, none of the procyanidins were determined nor were the cryptochlorogenic acid or p-coumaroylquinic acid from flavanols and hydroxycinnamic acids groups, respectively. As Khanizadeh et al. [3] described, the lack of substrate for PPO enzyme may be the cause of non-browning. This statement may be further supported by the results of PPO activity, where no statistically significant differences between our tested cultivars were determined. Following storage, the activity of PPO decreased in both cultivars. The low PPO activity is the main mechanism behind cv. 'Ambrosia' non-browning, but based on these results, it is rather the lack of substrate than the lower PPO activity that is the cause, therefore it is more similar to the 'Eden' cultivar's background. The content of dihydrochalcones differed between cultivars only following storage and the flavonol content varied between cultivars merely at L1. Both groups are known for their antioxidant role in apple [32,33], but the content of both groups in apple flesh is quite low. The POX activity was mostly influenced by time, and only the interaction between cultivar and location is statistically significant. With its various activities in plants, it is hard to make any assumptions without any further investigation. Nonetheless, the main differences between cultivars are well summarized by the heatmaps (Figures S1–S3).

Since the high phenolic content is preferred due to its beneficial role in human health [34], we also wanted to evaluate the apple peel from both cultivars. As done in flesh, the low sum of hydroxycinnamic acids and flavan-3-ols also stands out in the peel in cv. 'Majda'. There were no statistical differences in the contents of dihydrochalcones and flavonols between cultivars. Therefore, the peel has similar properties to flesh. This is not the best in the context of high antioxidant content. However, cv. 'Majda' contains anthocyanin cyanidin-3-O-galactoside. Anthocyanins are known for their beneficial role in human health [35].

With interesting results from flesh and peel, we wanted to broaden the knowledge on the phenolic content in cv. 'Majda', to see if this trait is present through the whole tree or if it is present just in the fruit. The group of flavonols and flavan-3-ols shows a similar pattern as flesh and peel, while the hydroxycinnamic acids and dihydrochalcones are even higher in cv. 'Majda' leaves in comparison to leaves of cv. 'Golden Delicious'. Both hydroxycinnamic acids and dihydrochalcones are known for their role as antioxidants and the role against various pathogens [32,36]. This might explain the overall lower sensitivity to pathogens of cv. 'Majda', which is an important factor for growers. This is encouraging, since in addition to the great fruit attributes, the sensitivity to diseases is a key concern for growers. Location also had an influence on the phenolic content, which was expected due to the different weather conditions.
