Antioxidant and hepatoprotective effects against acute CCl4 -induced liver damage in mice from red-fleshed apple flesh flavonoid extract.

Apple (Malus domestica Borkh.) is an important fruit tree species worldwide. Apple fruits are favored by consumers because of their antioxidative, anti-inflammatory, and antitumor effects as well as their protective effects against cardiovascular diseases and other health benefits. There is considerable interest in red-fleshed apple fruits among breeders because of their high flavonoid and anthocyanin contents. However, the flavonoids extracted from red-fleshed apple fruits must still be functionally characterized, especially regarding their protective effects against certain pathologies. In this study, the flavonoid components and contents in the extracts prepared from red-fleshed apple cultivar "Meihong" were determined. Additionally, the in vitro antioxidant activities and protective effects of the extracts against CCl4 -induced acute liver injury were investigated. The red-fleshed apple flesh flavonoid extract (RAFF) exhibited strong in vitro antioxidant activities. Compared with the model control mice treated with CCl4 , the mice pretreated with high (800 mg/kg·bw), middle (400 mg/kg·bw), and low (200 mg/kg·bw) RAFF doses had significantly lower CCl4 -induced serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities. Moreover, the RAFF pretreatment also significantly decreased the liver malondialdehyde activity and prevented the CCl4 -induced decrease in liver superoxide dismutase, glutathione peroxidase, catalase, and reduced glutathione levels. Furthermore, a histopathological examination revealed that RAFF inhibited the inflammatory cell infiltration and cell boundary loss caused by CCl4 in the liver. Thus, RAFF is a natural antioxidant with significant antioxidative activities and liver protective effects. The results of this study may be relevant for enhancing the application of the red-fleshed apple fruit extract as a food additive. PRACTICAL APPLICATION: We took the self-selected red-fleshed apple cultivar "Meihong" as the unique research material, and the active ingredients of its flavonoid extract, in vitro antioxidant activity and hepatoprotective effect were analyzed. It is of great significance to promote the development of the red-fleshed apple industry, and also provides an important reference for the development of natural antioxidants.

MdMYB6 regulates anthocyanin formation in apple both through direct inhibition of the biosynthesis pathway and through substrate removal.

Anthocyanin biosynthesis and sugar metabolism are important processes during plant growth, but the molecular interactions underlying these pathways are still unclear. In this work, we analyzed the anthocyanin and soluble sugar contents, as well as the transcript levels of transcription factors that are known to be related to the biosynthesis of anthocyanin in 'Hongcui 1' apple flesh during fruit development. Overexpression of MdMYB6 in red-fleshed calli was found to reduce anthocyanin content and result in downregulated expression of the MdANS and MdGSTF12 proteins. Yeast one-hybrid and electrophoretic mobility shift analyses showed that MdMYB6 could directly bind to the promoters of MdANS and MdGSTF12, indicating that MdMYB6 could inhibit anthocyanin biosynthesis by regulating MdANS and MdGSTF12. Overexpression of MdTMT1 in the Arabidopsis tmt1 mutant restored the glucose and fructose contents to the wild-type levels, while overexpression of MdTMT1 in red-fleshed calli increased the contents of glucose and fructose but reduced the contents of UDP-glucose, UDP-galactose, and anthocyanin. Using a GUS reporter system, yeast one-hybrid, chromatin immunoprecipitation-PCR and electrophoretic mobility shift analyses, we found that MdMYB6 could bind to the promoter of MdTMT1, resulting in increased promoter activity. Overexpression of MdMYB6 in calli overexpressing MdTMT1 increased the expression of MdTMT1, which led to reduced contents of UDP-glucose and UDP-galactose and decreased anthocyanin content compared to those of the calli that overexpressed MdTMT1. This finding suggested that MdMYB6 could also inhibit anthocyanin biosynthesis by regulating MdTMT1 to decrease the contents of UDP-glucose and UDP-galactose. Taken together, these results showed that MdMYB6 and MdTMT1 play key roles in both anthocyanin biosynthesis and sugar transport.

The R2R3-MYB transcription factor MdMYB24-like is involved in methyl jasmonate-induced anthocyanin biosynthesis in apple.

Anthocyanins in apple species are important secondary metabolites that are beneficial for human health. Previous studies revealed that methyl jasmonate (MeJA) promotes anthocyanin accumulation by up-regulating the transcription of related genes. In this study, we isolated a jasmonate (JA)-induced apple MYB gene, MdMYB24-like (MdMYB24L). The encoded nuclear protein contains a conserved R2R3 domain and is homologous to Arabidopsis thaliana AtMYB24. Additionally, MdMYB24L was observed to interact with JA signaling factors (MdJAZ8, MdJAZ11, and MdMYC2) in yeast and in planta. The MdMYC2 protein was also targeted by MdJAZ8 and MdJAZ11, which are rapidly degraded under MeJA treatment. The overexpression of MdMYB24L resulted in higher anthocyanin contents in the transgenic apple 'Orin' calli than in the wild-type control calli. Moreover, the expression levels of the anthocyanin biosynthesis structural genes MdUFGT and MdDFR were up-regulated in the transgenic calli. Furthermore, MdMYB24L positively regulated the transcription of MdDFR and MdUFGT by binding to the MYB-binding site motifs in their promoters. Interestingly, the interaction between MdMYC2 and MdMYB24L further enhanced the transcription of MdUFGT, whereas MdJAZ8 and MdJAZ11 attenuated this effect. We herein provide new details regarding the molecular mechanism by which MYB transcription factors help regulate anthocyanin biosynthesis via JA signaling pathways.

The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple.

KEY MESSAGE: The regulator MdERF1B in the apple (Malus × domestica) ethylene pathway mainly acts on MdMYB9 and MdMYB11 to regulate anthocyanin and proanthocyanidin accumulation. Dietary anthocyanins and proanthocyanidins (PAs) have health benefits for humans, and are associated with decreased risks of coronary heart disease and cancer. Ethylene can enhance reddening of apple (Malus × domestica), but the regulatory mechanism is poorly understood. In this study, an ethylene response factor (ERF), MdERF1B, was identified and functionally characterized. 'Orin' calli overexpressing MdERF1B were generated and then analyzed by quantitative reverse transcription-PCR. Compared with the control calli, the MdERF1B-overexpressing calli showed increased expression levels of MdACO1, MdERF1, and MdERF3 in the ethylene pathway and MdCHS, MdCHI, MdF3H, MdDFR, MdANS, MdLAR, MdANR, MdMYB9 and MdMYB11 in the flavonoid pathway. As a result, the levels of anthocyanins and PAs were significantly increased in the MdERF1B-overexpressing calli. MdERF1B interacted with MdMYB9, MdMYB1, and MdMYB11 proteins in yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays. Furthermore, in yeast one-hybrid and electrophoretic mobility shift assays, MdERF1B also bound to the promoters of MdMYB9, MdMYB1, and MdMYB11. In a luciferase reporter assay, MdERF1B mainly activated proMdMYB9 and proMdMYB11, promoting their expression levels. This was in agreement with MdERF1B's overexpression in calli, which barely affected MdMYB1 expression. Taken together, our findings provide an insight into the regulatory mechanisms in the ethylene pathway that increase anthocyanin and PA accumulation in apple.

Overexpression of a repressor MdMYB15L negatively regulates anthocyanin and cold tolerance in red-fleshed callus.

The cold-induced metabolic pathway and anthocyanin biosynthesis play important roles in plant growth. In this study, we identified a bHLH binding motif in the MdMYB15L protein using protein sequence analyses. Yeast two-hybrid and pull-down assays showed that MdMYB15L could interact with MdbHLH33. Overexpressing MdMYB15L in red-fleshed callus inhibited the expression of MdCBF2 and resulted in reduced cold tolerance but did not affect anthocyanin levels. Chip-PCR and EMSA analysis showed that MdMYB15L could bind the type II cis-acting element found in the MdCBF2 promoter. Overexpressing MdMYB15L in red-fleshed callus overexpressing MdbHLH33 also reduced cold tolerance and reduced MdbHLH33-induced anthocyanin biosynthesis. Knocking out the bHLH binding sequence of MdMYB15L (LBSMdMYB15L) prevented LBSMdMYB15L from interacting with MdbHLH33. Overexpressing LBSMdMYB15L in red-fleshed callus overexpressing MdbHLH33 also reduced cold tolerance and reduced MdbHLH33-induced anthocyanin biosynthesis. Together, these results suggested that an apple repressor MdMYB15L might play a key role in the cold signaling and anthocyanin metabolic pathways.