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Flavonoids play essential roles in human health. Apple (Malus domestica Borkh.), one of the most widely produced and economically important fruit crops in temperate regions, is a significant source of flavonoids in the human diet and is among the top nutritionally rated and most widely consumed fruits worldwide. Epidemiological studies have shown that the consumption of apples, which are rich in a variety of free and easily absorbable flavonoids, is associated with a decreased risk of various diseases. However, apple production is challenged by serious inbreeding problems. The narrowing of the hereditary base has resulted in apples with poor nutritional quality and low flavonoid contents. Recently, there have been advances in our understanding of the roles that Malus sieversii (Ledeb.) M.Roem has played in the process of apple domestication and breeding. In this study, we review the origin of cultivated apples and red-fleshed apples, and discuss the genetic diversity and construction of the core collections of M. sieversii. We also discuss current research progress and breeding programs on red-skinned and red-fleshed apples and summarize the exploitation and utilization of M. sieversii in the breeding of high-flavonoid, and red-fleshed apples. This study highlights a valuable pattern of horticultural crop breeding using wild germplasm resources. The future challenges and directions of research on the molecular mechanisms of flavonoid accumulation and high-flavonoid apple breeding are discussed., Molecular biology: Making apples even healthier They say that an apple a day keeps the doctor away, but the breeding of new varieties of red-fleshed apples with higher flavonoid contents could make this widely-consumed fruit even healthier. Apples are one of the richest dietary sources of flavonoids—metabolites credited with reducing the risk of various diseases—but during the process of domestication, the genetic diversity and nutritional quality of apples have decreased. In this review, Xue-Sen Chen at Shandong Agricultural University in China and colleagues track the evolution of modern cultivated apples from their ancestor, Malus sieversii, and outline recent developments in our understanding of the molecular pathways underpinning flavonoid synthesis. They also consider how M. sieversii could be used to create new varieties of tasty red-skinned and red-fleshed apples, enriched with beneficial metabolites.

Protoplast transient expression is a powerful strategy for gene functional characterization, especially in biochemical mechanism studies. We herein developed a highly efficient transient expression system for apple protoplasts. The abilities of the Arabidopsis thaliana and Malus domestica ubiquitin-10 (AtUBQ10 and MdUBQ10) promoters to drive the expression of multiple genes were compared with that of the CaMV 35S promoter, and the results revealed that the AtUBQ10 and MdUBQ10 promoters were more efficient in apple protoplasts. With this system, we demonstrated that active AtMKK7ac could activate MAPK6/3/4 signaling cascades, which further regulated MdWRKY33 phosphorylation and stability in apple. Furthermore, the ligand-induced interaction between the immune receptor AtFLS2 and the coreceptor AtBAK1 was reconstituted in apple protoplasts. We also found that the stability of the bacterial effector AvrRpt2 was regulated by feedback involving auxin and the immune regulator RIN4. The system established herein will serve as a useful tool for the molecular and biochemical analyses of apple genes.

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.

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.

Auxin signaling, which is crucial for normal plant growth and development, mainly depends on ARF–Aux/IAA interactions. However, little is known regarding the regulatory effects of auxin signaling on anthocyanin metabolism in apple (Malus domestica). We investigated the functions of MdARF13, which contains a repression domain and is localized to the nucleus. This protein was observed to interact with the Aux/IAA repressor, MdIAA121, through its C-terminal dimerization domain. Protein degradation experiments proved that MdIAA121 is an unstable protein that is degraded by the 26S proteasome. Additionally, MdIAA121 stability is affected by the application of exogenous auxin. Furthermore, the overexpression of MdIAA121 and MdARF13 in transgenic red-fleshed apple calli weakened the inhibitory effect of MdARF13 on anthocyanin biosynthesis. These results indicate that the degradation of MdIAA121 induced by auxin treatment can release MdARF13, which acts as a negative regulator of the anthocyanin metabolic pathway. Additionally, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays confirmed that MdMYB10 interacts with MdARF13. A subsequent electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that MdARF13 directly binds to the promoter of MdDFR, which is an anthocyanin pathway structural gene. Interestingly, chromatin immunoprecipitation–quantitative real-time PCR results indicated that the overexpression of MdIAA121 clearly inhibits the recruitment of MdARF13 to the MdDFR promoter. Our findings further characterized the mechanism underlying the regulation of anthocyanin biosynthesis via Aux/IAA–ARF signaling., A green light for red apples Insights into a hormonal signaling pathway controlling apple coloration could help growers to consistently produce eye-catching fruit. Auxins are signaling molecules that regulate many important metabolic pathways in plants. Research from a team led by Xue-sen Chen at Shandong Agricultural University in China now reveals how the auxin naphthalene acetic acid (NAA) controls production of anthocyanin, the molecule that gives red apples their distinctive coloration. The researchers determined that increasing NAA levels result in reduced production of anthocyanin. These effects are mediated by a subset of auxin-associated proteins that regulate the expression of genes that contribute to the synthesis of this pigment. The color of fruit can strongly influence consumer purchasing decisions, and this signaling pathway could help horticulturalists to ensure that apples retain their visual appeal even under inclement growing conditions.