THE ANTHOCYANIN BIOSYNTHETIC REGULATOR MDMYB1 POSITIVELY REGULATES ASCORBIC ACID BIOSYNTHESIS IN APPLE

Ascorbic acid (AsA, vitamin C) is involved in the regulation of many aspects of plant growth and development. It is an essential micronutrient for humans and can prevent scurvy, maintain the health of gums and blood vessels, reduce the level of plasma cholesterol and enhance the immune systen. Apple cultivars Orin and Guanghui were crossed to obtain a group of hybrid offspring with and without red flesh in the course of assessing apple germplasm resources. Unexpectedly, the red-flesh apples had higher AsA contents than other apples. Further studies showed that the anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA. This finding reveals the mechanism leading to high AsA levels in red-flesh apples and suggests a new idea to cultivate red-flesh apples with high AsA contents and produce AsA efficiently and without pollution.

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Ascorbic acid (AsA) is an important antioxidant and has an essential role in plant resistance to oxidative stress [1] . In addition to its ability to scavenge reactive oxygen species (ROS), AsA is involved in regulating many aspects of plant growth and development, including seed germination, flower induction, photosynthesis, fruit development, senescence and stress tolerance [1][2][3][4][5][6][7] . The biosynthesis of AsA occurs mainly through four pathways, namely the D-glucosone, D-galacturonate, myo-inositol and D-mannose/Lgalactose pathways [8][9][10][11][12] . The D-mannose/L-galactose synthesis pathway is particulary important in AsA biosynthesis. The biosynthesis of AsA is influenced by different stages of plant development (such as germination, fruit development, and senescence) and external light conditions [2,13] . However, the molecular mechanism by which the environmental factors regulate the synthesis of AsA remains unclear. AsA is an essential micronutrient for humans and can prevent scurvy, maintain the health of gums and blood vessels, reduce the level of plasma cholesterol and enhance the immune system. However, high cost, waste of resources and environmental pollution in AsA production have restricted the development of industrial AsA production. In the course of assessing apple germplasm resources, we crossed apple cultivars Orin and Guanghui to obtain a group of hybrid offspring with and without red flesh ( Fig. 1(a)) [14] . The red-flesh apples contained large amounts of visible anthocyanin ( Fig. 1(a)). In addition, they unexpectedly had higher AsA contents than other apples ( Fig. 1(b)). This finding prompted us to explore the mechanism leading to high AsA levels in red-flesh apples.
AsA will be synthesized in large quantities as reactive oxygen scavengers when plants are under stress and excessive ROS are produced [1] . AsA is oxidized to dehydroascorbic acid (DHA) by ascorbic acid oxidase in the process of scavenging ROS. Also, the enzyme dehydroascorbic acid reductase (DHAR) catalyzes the formation of AsA from DHA. The AsA-DHA cycle has an important role in maintaining the dynamic balance of AsA and regulating plant growth and stress response [1,13,15] . Here, DHA contents and DHAR enzyme activity in red-flesh and other apples were also determined. Red-flesh apples had lower DHA contents and higher DHAR enzyme than other apples ( Fig. 1(c,d)). Also, quantitative real-time (qRT)-PCR shows that the expression level of the MdMYB1 gene (GenBank accession number: MDP0000259614) was positively correlated with the expression level of MdDHAR (GenBank accession number: MDP0000175246), that is, the expression levels of MdMYB1 and MdDHAR in red-flesh apples were higher than in other apples ( Fig. 1(e)). These results indicate a possible correlation between anthocyanins and AsA.
Anthocyanins are important secondary metabolites in addition to AsA and also have an important role in plant growth and development [16] . At the transcriptional level, anthocyanin biosynthesis is regulated by the MYB-bHLH-WD40 protein complex [17] and the MYB transcription factor has a central role. MdMYB1 and its alleles (MdMYB10 and MdMYBA) have been shown to be key positive regulators of anthocyanin biosynthesis in apples [18][19][20] . The red flesh in apples is caused by the overexpression of MdMYB1 (MdMYB10) and we therefore obtained MdMYB1-overexpressing apple callus in order to determine the correlation between anthocyanin and AsA ( Fig. 1(f)). As expectated, the contents of AsA in MdMYB1green fluorescent protein (GFP) transgenic apple callus were higher than in the control (GFP), while the contents of DHA were lower than in the control (GFP) ( Fig. 1(g,h)). DHAR enzyme activity and MdDHAR gene expression level in MdMYB1-GFP transgenic apple callus were higher than in the control (GFP) ( Fig. 1(i,j)). These results indicate that overexpression of the MdMYB1 gene may increase the activity of the DHAR enzyme by increasing the expression of the MdDHAR gene, thereby promoting the conversion of DHA into AsA, and finally increasing the contents of AsA. In addition, we found that overexpression of MdMYB1 in apple callus also promoted the expression of the ascorbate oxidase gene MdAO (GenBank accession number: XP_028958650.1), the ascorbate peroxidase gene MdAPX1 (GenBank accession number: MDP0000210077) and the monodehydroascorbate reductase gene MdMDHAR (GenBank accession number: MDP0000199989) ( Fig. 1(j)), suggesting that MdMYB1 may play a key role in AsA-DHA homeostasis.
We analyzed the promoter sequence of MdDHAR in order to further reveal the transcriptional regulation mechanism of MdMYB1 on the MdDHAR gene. The MdDHAR promoter sequence was found to contain a binding site (P1) for the MYB transcription factor. Chromatin immunoprecipitation (ChIP)-PCR assays were conducted to determine the binding of MdMYB1 to the promoter of MdDHAR. MdMYB1 protein precipitated from MdMYB1-GFP transgenic callus and enrichment of MdDHAR promoter sequence detected by qRT-PCR. This indicates that the enrichment of the P1 region was higher than the control region (P2, Fig. 1(k)), and that MdMYB1 may directly bind to the P1 region of the MdDHAR promoter. We also conducted electrophoretic mobility shift assays to verify the interaction between MdMYB1 and the MdDHAR promoter. As shown in Fig. 1(l), MdMYB1 directly bound to the 5′-CTGTTG-3′ site of the MdDHAR promoter, while MdMYB1 did not bind when the 5′-CTGTTG-3′ site was mutated to 5′-CGGTGG-3′. These data indicate that MdMYB1 binds to the MdDHAR promoter.
To study the transcriptional regulation function of MdMYB1 on MdDHAR, the promoter sequence of MdDHAR was cloned to the pCAMBIA1391-GUS vector and transformed into the apple callus ( Fig.  1(m)). GUS activity detection results show that compared with MdDHAR-GUS callus alone, MdMYB1 overexpressed on MdDHAR-GUS basis significantly increased its GUS activity ( Fig. 1(n)). In addition, the promoter sequence of MdDHAR was cloned to the pGreen0800-LUC vector and full length MdMYB1 was inserted into the pGreen62-SK vector (Fig. 1(o)). The recombinant plasmids were transformed into Agrobacterium tumefaciens and injected into tobacco leaves. The fluorescence detection results show that MdMYB1 overexpression significantly increased the fluorescence activity of MdDHAR ( Fig. 1(p)). These results suggest that MdMYB1 activates the expression of MdDHAR by directly binding to its promoter. Anthocyanin and AsA play similar biological roles in plant growth and development [1,16] . The crosstalk between them has never been revealed. Here, we found that apples with high anthocyanin content contained more AsA, suggesting that there may be a positive correlation between anthocyanin and AsA. We hypothesized that anthocyanin biosynthesis might disrupt AsA-DHA homeostasis. The biological function of anthocyanin may also require the coordination of AsA. Of course, the relationship between anthocyanin and AsA needs further study.
In summary, our studies reveal why the AsA contents of red-flesh apples are higher than those of other apples. This is because excessive MdMYB1 breaks the homeostasis of AsA and DHA in apple fruits, and MdMYB1 directly activates the expression of the dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA (Fig. 1(q)). This finding reveals the mechanism leading to high AsA levels in red-flesh apples and provides new information that is useful in the cultivatation of red-flesh apples with high AsA contents to produce AsA efficiently and without pollution.