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Anthocyanins, a major class of compounds derived from the flavonoid pathway, are important pigments of apple fruit. They can also prevent certain diseases and are beneficial to human health. Fruit pigmentation is a key quality trait that influences consumer preference; therefore, it is of great importance to investigate its regulatory mechanism. Here, we identified a MYB transcription factor (TF), MdMYB114, whose transcript level increased in the skin of the deep red apple fruit. It was determined to belong to the R2R3-MYB TF family and was localized in the nucleus. MdMYB114 overexpression led to anthocyanin accumulation in apple calli. MdMYB114 was not able to form an MBW complex but could enhance anthocyanin biosynthesis and transport by directly binding to the promoters of MdANS, MdUFGT, and MdGST to promote their expression. In addition, multiple assays revealed that MdbZIP4-like, a basic leucine-zipper TF, could directly bind to the MdMYB114 promoter to enhance its expression. Taken collectively, our results provide evidence that MdMYB114 is a positive regulator of anthocyanin biosynthesis and transport and it functions downstream of MdbZIP4-like in apple fruit., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Anthocyanin and proanthocyanidin (PA) play important roles in plant growth and development. Although previous studies have identified many of the transcription factors involved in the anthocyanin and PA pathway, the regulation mechanisms of these pathways remain poorly understood. In this study, we identified a NAC transcription factor, MdNAC52, whose gene transcript levels increased during apple coloration. Apple calli overexpressing MdNAC52 accumulated anthocyanin. Yeast one-hybrid, electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that MdNAC52 could interact with the promoters of MdMYB9 and MdMYB11 to regulate anthocyanin biosynthesis. MdNAC52 was targeted by MdHY5 in response to light. Interestingly, MdNAC52 participated in the regulation of PA biosynthesis through controlling the expression of MdMYB9 and MdMYB11. MdNAC52 could also bind to the LAR promoter to regulate its expression and promote PA synthesis. Overall, these findings establish that MdNAC52 binds to the promoters of MdMYB9 and MdMYB11 to promote anthocyanin and PA biosynthesis and directly regulates LAR to modulate PA metabolism. Our study provides new insights into the roles of a NAC transcription factor in regulating anthocyanin and PA accumulation in apple., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In plants, flavonoids are important secondary metabolites that contribute to the nutritional quality of many foods. Apple is a popular and frequently consumed food because of its high flavonoid content. In this study, flavonoid composition and content were detected and compared between red- and white-fleshed apples in a BC 1 hybrid population using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Transcriptomic analysis of the red- and white-fleshed apples was then performed using RNA-seq technology. By screening differentially expressed genes encoding transcription factors, we unearthed a WRKY-family transcription factor designated MdWRKY11. Overexpression of MdWRKY11 promoted the expression of F3H, FLS, DFR, ANS, and UFGT and increased the accumulation of flavonoids and anthocyanin in apple calli. Our findings explored the novel role of MdWRKY11 in flavonoid biosynthesis and suggest several other genes that may also be potentially involved. This provides valuable information on flavonoid synthesis for the breeding of elite red-fleshed apples.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

Nitrogen limitation stress is known to markedly promote anthocyanin accumulation and upregulate anthocyanin biosynthesis-related genes; however, the underlying molecular mechanisms have not been fully characterized. In this study, a callus was induced from the leaves of an R6 homozygous red-fleshed apple, the hybrid offspring of Malus sieversii f. niedzwetzkyana and Malus × domestica ‘Fuji.’ We analyzed the growth and accumulation of anthocyanin in callus under different nitrogen concentrations (0.01, 0.02, 0.04, and 0.06 M NO3−). The results showed that the callus growth in the 0.01 M NO3− condition was minimal, but its anthocyanin content was significantly elevated compared with those under the other nitrogen treatments. As the concentration of nitrogen increased from 0.01 to 0.04 M, the expression of the anthocyanin structural genes, MdCHS and MdDFR, and the transcription factor genes MdMYB9, MdMYB10, MdbHLH3, and MdbHLH33 were downregulated. On the contrary, the transcript levels of MdLOB52 and MdARF19 were increased by the nitrogen treatments. The overexpression of MdLOB52 inhibited anthocyanin accumulation in the callus. Subsequent yeast one-hybrid assays showed that MdARF19 can directly bind the promoter of MdLOB52, which may strengthen the activation of MdLOB52 and affect anthocyanin accumulation. Our findings provide new insights into the mechanisms by which the LOB genes respond to nitrogen to regulate anthocyanin biosynthesis in a red-fleshed apple callus. [ABSTRACT FROM AUTHOR]

Anthocyanins are biosynthesized on the cytosolic surface of the endoplasmic reticulum and then transported into the vacuole for storage. Glutathione S-transferases (GSTs) are considered to be responsible for the transport of anthocyanins into the vacuole. However, the regulatory mechanisms of GSTs in plants are still unclear. Here, we performed a genome-wide analysis and identified 69 GST genes in apple. The expression of MdGSTF6 was positively correlated with the anthocyanin content (r = 0.949) during 'Yanfu 8' fruit development. The overexpression of MdGSTF6 in the Arabidopsis thaliana tt19 mutant resulted in seedlings of 35S::MdGSTF6-GFP/tt19 that could accumulate anthocyanin and rescue its phenotype, suggesting that MdGSTF6 was an anthocyanin transporter. The silencing of MdGSTF6 affected anthocyanin accumulation in apple fruit. Moreover, the knockdown of MdGSTF6 by RNA interference in cultured 'Gala' seedlings inhibited anthocyanin accumulation. The interaction experiments showed that MdMYB1 could bind directly to the MdGSTF6 promoter to transcriptionally activate its expression. Collectively, our results demonstrate that MdGSTF6 encodes an important GST transporter of anthocyanins in apple fruit and provide evidence for the associated regulatory mechanisms. Therefore, MdMYB1 can not only regulate anthocyanin synthesis, but also control the transport of anthocyanin in apples. This information may be useful for further clarifying the regulation of anthocyanin transport in apple. Intracellular transport: Identifying how apple pigment is sent for storage The mystery of how pigmenting anthocyanins are transported to intracellular storage is starting to be revealed. Alongside giving fruit their color, anthocyanins confer benefits to plant and human health. Glutathione S-transferases (GSTs) are responsible for transporting anthocyanins to storage vacuoles, yet research is lacking on how GSTs are regulated. Xuesen Chen, from China's Shandong Agricultural University, and his team analyzed the activity of 23 GST genes in apple, finding one, MdGSTF6, was most highly active in the fruit-coloration stage of apple development. MdGSTF6 expression correlated with fruit anthocyanin levels and also restored anthocyanin levels in transport-suppressed plants, confirming its function. Chen's team then discovered that MdGSTF6 expression is activated by the protein MdMYB1. The MdMYB1 gene is the major regulatory gene in anthocyanin synthesis, and is now revealed to also influence their transport. [ABSTRACT FROM AUTHOR]