Phenolic Metabolism Of Red-fleshed Apples And Its Response To Stress

Phenolic compounds have strong antioxidant properties that enable them to protect the human body against cellular oxidation reactions. Apple is one of the fruit-tree species with abundant phenolic compounds. Apples with red fruit flesh are desirable for breeding. Red-fleshed apples are receiving increased attention because of their remarkable anthocyanin concentrations. To provide sufficient experimental evidence of their phenolic composition and strong antioxidant capacity, we evaluated and compared red-fleshed varieties and white-fleshed varieties. To obtain experimental evidence about the molecular mechanism for this pigmentation, we investigated how the practice of bagging and different developing stages might affect levels of phenolic compounds as well as the expression of anthocyanin biosynthetic and regulatory genes. Meanwhile, to provide basis for relationship research between phenolics and stress tolerance, we investigated the effects of abiotic deficit(deficit, salt, cold, heat et al.) on the levels of phenolic compounds as well as the expression of phenolic biosynthetic genes of the apple leaves.The main results are as follows:1. Concentrations of phenolics(i.e., total phenolics, flavonoids, flavanols, and anthocyanins) and antioxidant activity were significantly higher in flesh samples from red-fleshed apple varieties than from white-fleshed varieties. Flavonoid profiles also differed between the red- and white-fleshed varieties. The red-fleshed varieties was dominated by flavanols and anthocyanins, while the white-fleshed apple varieties was dominated by flavanols and flavonols. Among the red-fleshed varieties, ‘Roberts Crab’ had the highest concentrations of total phenolics, flavonoids, and anthocyanins, as well as the strongest antioxidant activity, while ‘Xiahongrou’ had the lowest levels of total phenolics and flavonoids and the weakest activity. HPLC analysis of those red-fleshed varieties revealed that individual phenolic compounds varied significantly between varieties. ‘Roberts Crab’ possessed the highest concentration of cyanidin-3-galactoside, phloridzin, epicatechin, catechin, quercetin-3-glucoside, whereas ‘Hongrouguo’ possessed the highest concentration of chlorogenic acid, procyanidin B1, procyanidin B2 in both apple portions. Correlation analyses showed that the concentration of total anthocyanins were highly correlated with antioxidant activity. All of these results suggest that red-fleshed apples are a promising source of favorable antioxidants for human nutrition.2. Differences in the rule of flesh anthocyanin accumulation were observed between two red-fleshed varieties. The anthocyanin content of ‘Xiahongrou’ flesh showed a declined pattern, while the anthocyanin content of ‘No.1 Hongxun’ flesh displayed an opposite tendency. For all the anthocyanin biosynthetic genes assayed, transcript levels in red-fleshed apples were significantly higher during all stages of fruit development than the levels found in white-fleshed varieties, especially in the flesh. The strong correlation between the expression of MYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit. Transcript levels of b HLH3 and b HLH33 were also assayed but did not appear to follow the pattern displayed for the biosynthetic genes, or for MYB10, with more consistent levels of expression throughout the developmental series and in both cultivars. These results confirm the previous findings that it is the MYB component from the proposed regulatory complex of MYB–b HLH–WD40 that is primarily responsible for the level of anthocyanin accumulation.3. For our flesh samples, bagging led to a decrease in anthocyanin concentrations, which were then partially recovered after those bags were removed. Except for F3 H in ‘No.1 Hongxun’, transcription levels for most anthocyanin biosynthetic genes in the flesh were enhanced after bag removal. MYB10 is a key transcriptional factor in the anthocyanin pathway of these two apple varieties, particularly in the flesh. These results suggest that the development of red coloring when bags are removed could be a result of light-induced coordinated expression by MYB10 and anthocyanin biosynthetic genes in red-fleshed apples.4. Our results indicate that the red-fleshed varieties are more tolerant to water stress, registering greater photosynthetic rate and leaf relative water content, associated with high antioxidant activity. Long-term deficit significantly decreased the content of anthocyanins in red-fleshed apple leaves. Meanwhile, the content of total phenolics, phloridzins and flavonols were also decreased. Moreover, most of genes involved in anthocyanin biosynthesis were down-regulated, whereas HCT, FLS, MYB10 were up-regulated. However, under short-term deficit condition, the content of anthocyanins, phloridzins and flavonols increased firstly and then decreased. Most of genes involved in anthocyanin biosynthesis were up-regulated. We proposed that the degradation induced by the drought stress is partly responsible for the changes in content of phenolics.5. Stress treatment significantly altered the phenolic compound concentrations, some of which were significantly increased. Anthocyanins, procyanidin B1 and chlorogenic acid were the most sensitive to much of stress, except heat stress. The concentration of phloridzin and flavonols were decreased. The expression of most of the phenolic biosynthetic genes was enhanced, with CHS being the most prominent. We proposed that CHS maybe primarily responsible for the response to abiotic stress.