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MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red-flesh phenotype due to a minisatellite insertion in its R 6 promoter, but R 6 :MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull-down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB-bHLH-WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163-bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R 6 by structural variation analysis, contains a typical W-box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R 6 background. These findings reveal a novel regulatory role of the WRKY-containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Enhancing fruit sugar contents, especially for high-flavonoid apples with a sour taste, is one of the main goals of horticultural crop breeders. This study analyzed sugar accumulation and the underlying mechanisms in the F2 progenies of a hybridization between the high-sugar apple (Malus × domestica) variety "Gala" and high-flavonoid apple germplasm "CSR6R6". We revealed that MdSWEET9b (sugars will eventually be exported transporter) helps mediate sugar accumulation in fruits. Functional characterization of MdSWEET9b in yeast mutants lacking sugar transport as well as in overexpressing and CRISPR/Cas9 knockdown apple calli revealed MdSWEET9b could transport sucrose specifically, ultimately promoting normal yeast growth and accumulation of total sugar contents. Moreover, MdWRKY9 bound to the MdSWEET9b promoter and regulated its activity, which responded to abscisic acid (ABA) signaling. Furthermore, MdWRKY9 interacted with MdbZIP23 (basic leucine zipper) and MdbZIP46, key ABA signal transducers, at the protein and DNA levels to enhance its regulatory effect on MdSWEET9b expression, thereby influencing sugar accumulation. Based on the contents of ABA in lines with differing sugar contents and the effects of ABA treatments on fruits and calli, we revealed ABA as one of the main factors responsible for the diversity in apple fruit sugar content. The results of this study have clarified how MdSWEET9b influences fruit sugar accumulation, while also further elucidating the regulatory effects of the ABA-signaling network on fruit sugar accumulation. This work provides a basis for future explorations of the crosstalk between hormone and sugar metabolism pathways., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The anthocyanin content is an important indicator of the nutritional value of most fruits, including apple (Malus domestica). Anthocyanin synthesis is coordinately regulated by light and various phytohormones. In this study on apple, we revealed the antagonistic relationship between light and brassinosteroid (BR) signaling pathways, which is mediated by BRASSINAZOLE-RESISTANT 1 (MdBZR1) and the B-box protein MdCOL6. The exogenous application of brassinolide inhibited the high-light-induced anthocyanin accumulation in red-fleshed apple seedlings, whereas increases in the light intensity decreased the endogenous BR content. The overexpression of MdBZR1 inhibited the anthocyanin synthesis in apple plants. An exposure to a high-light intensity induced the degradation of dephosphorylated MdBZR1, resulting in functional impairment. MdBZR1 was identified as an upstream repressor of MdCOL6, which promotes anthocyanin synthesis in apple plants. Furthermore, MdBZR1 interacts with MdCOL6 to attenuate its ability to activate MdUFGT and MdANS transcription. Thus, MdBZR1 negatively regulates MdCOL6-mediated anthocyanin accumulation. Our study findings have clarified the molecular basis of the integration of light and BR signals during the regulation of anthocyanin biosynthesis, which is an important process influencing fruit quality., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Sugar and anthocyanin are important indicators of fruit quality, and understanding the mechanism underlying their accumulation is essential for breeding high-quality fruit. We identified an R2R3-MYB transcription factor MdMYB305 in the red-fleshed apple progeny, which was positively correlated with fruit sugar content but negatively correlated with anthocyanin content. Transient injection, stable expression [overexpressing and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)], and heterologous transformation of tomato confirmed that MdMYB305 promotes the accumulation of sugar and inhibits the synthesis of anthocyanin. A series of molecular experiments (such as electrophoretic mobility shift and luciferase assays) confirmed that MdMYB305 combines with sugar-related genes (MdCWI1/MdVGT3/MdTMT2) and anthocyanin-related genes (MdF3H/MdDFR/MdUFGT), promoting and inhibiting their activities, and finally regulating the sugar and anthocyanin content of fruits. In addition, the study also found that MdMYB305 competes with MdMYB10 for the MdbHLH33 binding site to balance sugar and anthocyanin accumulation in the fruits, which provides a reference value for exploring more functions of the MYB-bHLH-MYB complex and the balance relationship between sugar and anthocyanin in the future., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Drought is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple ( Malus domestica ). Heat shock factors (HSFs) have well-documented functions in stress responses, but their roles in flavonoid synthesis and the flavonoid-mediated drought response mechanism remain elusive. In this study, we demonstrated that a drought-responsive HSF, designated MdHSFA8a, promotes the accumulation of flavonoids, scavenging of reactive oxygen species, and plant survival under drought conditions. A chaperone, HEAT SHOCK PROTEIN90 (HSP90), interacted with MdHSFA8a to inhibit its binding activity and transcriptional activation. However, under drought stress, the MdHSP90-MdHSFA8a complex dissociated and the released MdHSFA8a further interacted with the APETALA2/ETHYLENE RESPONSIVE FACTOR family transcription factor RELATED TO AP2.12 to activate downstream gene activity. In addition, we demonstrated that MdHSFA8a participates in abscisic acid-induced stomatal closure and promotes the expression of abscisic acid signaling-related genes. Collectively, these findings provide insight into the mechanism by which stress-inducible MdHSFA8a modulates flavonoid synthesis to regulate drought tolerance., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Salt stress is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus × domestica). In this study, we demonstrate that a salt-responsive MYB transcription factor (TF), designated as MdMYB63, promotes survival under salt stress. Overexpression of MdMYB63 in apple calli significantly enhanced salt tolerance. Screening of the AP2/ERF family of TFs identified MdERF106 as an interaction partner of MdMYB63. Further analyses showed that the MdMYB63-MdERF106 complex significantly promotes the expression of downstream MdSOS1, thereby improving the Na + expulsion and salt tolerance of apple. These functional analyses of MdMYB63 have provided valuable insights into the regulatory network of salt tolerance, and lay a theoretical foundation for the cultivation of new salt-tolerant apple varieties., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: Saline, alkaline, and saline-alkaline stress severely affect plant growth and development. The tolerance of plants to these stressors has long been important breeding objectives, especially for woody perennials like apple. The aims of this study were to identify quantitative trait loci (QTLs) and to develop genomics-assisted prediction models for salt, alkali, and salt-alkali tolerance in apple rootstock., Results: A total of 3258 hybrids derived from the apple rootstock cultivars 'Baleng Crab' (Malus robusta Rehd., tolerant) × 'M9' (M. pumila Mill., sensitive) were used to identify 17, 13, and two QTLs for injury indices of salt, alkali, and salt-alkali stress via bulked segregant analysis. The genotype effects of single nucleotide polymorphism (SNP) markers designed on candidate genes in each QTL interval were estimated. The genomic predicted value of an individual hybrid was calculated by adding the sum of all marker genotype effects to the mean phenotype value of the population. The prediction accuracy was 0.6569, 0.6695, and 0.5834 for injury indices of salt, alkali, and salt-alkali stress, respectively. SNP182G on MdRGLG3, which changes a leucine to an arginine at the vWFA-domain, conferred tolerance to salt, alkali, and salt-alkali stress. SNP761A on MdKCAB, affecting the Kv_beta domain that cooperated with the linked allelic variation SNP11, contributed to salt, alkali, and salt-alkali tolerance in apple rootstock., Conclusions: The genomics-assisted prediction models can potentially be used in breeding saline, alkaline, and saline-alkaline tolerant apple rootstocks. The QTLs and the functional markers may provide insight for future studies into the genetic variation of plant abiotic stress tolerance.

Methylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured 'Fuji' apples and found that differences in the transcript levels of MdMYB1, which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (-1246 to -780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA-directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4-binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Ultraviolet-B (UV-B) radiation promotes anthocyanin synthesis in many plants. Although several transcription factors promote anthocyanin synthesis in response to UV-B radiation, the underlying mechanism remains unclear. In this study, the MdWRKY72 transcription factor gene was isolated from the 'Taishanzaoxia' apple genome. Quantitative real-time PCR analyses revealed that the genes encoding enzymes and transcription factors involved in the anthocyanin synthesis pathway (MdANS, MdDFR, MdUFGT, and MdMYB1) were more highly expressed in MdWRKY72-overexpressing transgenic calli than in the wild-type 'Orin' apple calli. The results indicated that MdWRKY72 increases anthocyanin synthesis in transgenic calli exposed to UV-B radiation. The results of a gel shift assay and chromatin immunoprecipitation proved that MdWRKY72 promotes MdMYB1 expression indirectly by binding to a W-box element in the MdHY5 promoter and directly by binding to a W-box element in the MdMYB1 promoter. Thus, MdWRKY72 increases anthocyanin synthesis via direct and indirect mechanisms. These findings may be useful for elucidating the molecular mechanism underlying UV-B-induced anthocyanin synthesis mediated by MdWRKY72., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Red-fleshed apples are popular as a result of their high anthocyanin content. MdMYB10 and its homologues are known to be important regulators of anthocyanin synthesis in apple, but the roles of other transcription factors are not well-understood. Here, we explored the role of MdWRKY11 in regulating anthocyanin synthesis in apple flesh. Overexpression of MdWRKY11 in apple callus could significantly promote anthocyanin accumulation, and the expression of some MYB transcription factors and structural genes increased significantly. In binding analyses, MdWRKY11 bound to W-box cis -elements in the promoters of MdMYB10 , MdMYB11 , and MdUFGT . However, MdWRKY11 did not interact with MdMYB10, MdbHLH3, or MdWD40 proteins, the members of the MBW complex. Sequence analyses revealed that another W-box cis -element was present in the promoter of MdHY5 (encoding a photoresponse factor), and MdWRKY11 was able to bind to the promoter of MdHY5 and promote its activity. Our findings clarify the role of MdWRKY11 in anthocyanin synthesis in red-fleshed apple and imply that other novel genes may be involved in anthocyanin synthesis.

Ultraviolet-B (UV-B) radiation and low temperature promote the accumulation of anthocyanins, which give apple skins their red colour. Although many transcription regulators have been characterized in the UV-B and low-temperature pathways, their interregulation and synergistic effects are not well understood. Here, a B-box transcription factor gene, MdBBX20, was characterized in apple and identified to promote anthocyanin biosynthesis under UV-B conditions in field experiments and when overexpressed in transgenic apple calli. The transcript level of MdBBX20 was significantly induced by UV-B. Specific G-box elements in the promoters of target genes were identified as interaction sites for MdBBX20. Further experimental interrogation of the UV-B signalling pathways showed that MdBBX20 could interact with MdHY5 in vitro and in vivo and that this interaction was required to significantly enhance the promoter activity of MdMYB1. MdBBX20 also responded to low temperature (14°C) with the participation of MdbHLH3, which directly bound a low temperature-response cis elements in the MdBBX20 promoter. These findings demonstrate the molecular mechanism by which MdBBX20 integrates low-temperature- and UV-B-induced anthocyanin accumulation in apple skin., (© 2019 John Wiley & Sons Ltd.)

Light is an important environmental factor affecting plant growth and development. Additionally, HY5 is a central factor that coordinates light signal transduction and regulates the expression of flower color-related genes. However, there are few reports describing the co-regulation of apple fruit coloration by MdHY5 and MYB transcription factors. In this study, we detected a light-inducible gene, MdMYBDL1, which encodes a MYB-like domain and is homologous to AtMYBD in Arabidopsis thaliana. Moreover, we observed that MdHY5 binds to the G-box element of the MdMYBDL1 promoter to upregulate expression. The overexpression of MdMYBDL1 enhanced anthocyanin accumulation in apple calli and inhibited the expression of MdMYB16 and its homolog, MdMYB308. Furthermore, MdMYB16 can form a dimer with MdMYB308 and functions as a negative regulator of anthocyanin biosynthesis. Interestingly, MdMYB16 and MdMYB308 promoter activities were inhibited by MdMYBDL1 and MdHY5. These findings imply that MdHY5 responds to light signals and functions upstream of different types of MYB transcription factors, ultimately regulating anthocyanin accumulation in apples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In many plants, anthocyanin biosynthesis is affected by environmental conditions. Ultraviolet-B (UV-B) radiation promotes anthocyanin accumulation and fruit coloration in apple skin, whereas high temperature suppresses these processes. In this study, we characterized a B-box transcription factor, MdCOL4, from 'Fuji' apple, and identified its role in anthocyanin biosynthesis by overexpressing its encoding gene in apple red callus. The expression of MdCOL4 was reduced by UV-B, but promoted by high temperature. We explored the regulatory relationship between heat shock transcription factors (HSFs) and MdCOL4, and found that MdHSF3b and MdHSF4a directly bound to the heat shock element cis-element of the MdCOL4 promoter. MdCOL4 interacted with MdHY5 to synergistically inhibit the expression of MdMYB1, and MdCOL4 directly bound to the promoters of MdANS and MdUFGT, which encode genes in the anthocyanin biosynthetic pathway, to suppress their expression. Our findings shed light on the molecular mechanism by which MdCOL4 suppresses anthocyanin accumulation in apple skin under UV-B and high temperature., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Anthocyanin biosynthesis is induced by cytokinins, and is regulated by MYB transcription factors. However, the underlying molecular mechanisms have not been fully characterised. In the present study, red-fleshed apple callus were induced from the leaves of an R6/R6 homozygous line, which was the hybrid offspring of Malus sieversii f. niedzwetzkyana and 'Fuji'. We analysed the callus anthocyanin contents in response to different cytokinin concentrations. We observed that cytokinin treatments upregulated the expression of anthocyanin structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3. Additionally, the expression of MdMYBL2, which encodes the bHLH and EAR motifs, was inhibited by cytokinin treatments. The MdMYBL2-overexpressing callus had lower anthocyanin contents than the wild-type controls. We noted that the expression levels of anthocyanin biosynthesis structural genes MdDFR and MdUFGT and transcription factor genes MdMYB10 and MdbHLH3 were strongly suppressed in the transgenic callus. Subsequent yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays indicated that MdMYBL2 interacts with MdbHLH3, which may influence the expression of anthocyanin biosynthesis-related genes. Our findings may provide new insights into how MYB transcription factors influence the cytokinin-regulated anthocyanin biosynthesis in red-fleshed apples.

The transition from vegetative to reproductive growth is the most dramatic phase change in plants. To better understand the molecular regulation of floral transition and flower development in red-fleshed apple (Malus sieversii f. niedzwetzkyana), we isolated and characterized a floral MADS-box gene, MdAGL24-like, which shares sequence similarity with AGAMOUS-LIKE 24 (AGL24) from other species. Spatial expression analysis showed that MdAGL24-like dynamically expressed in flowers, followed by roots and fruits. Subcellular localization analysis indicated that, like other transcript factors, MdAGL24-like was localized in the nucleus. Protein interaction analysis showed that MdAGL24-like could interact with MdSOC1 and MdAP1 in vivo and in vitro. MdAGL24-like and MdSOC1 could increase each other's expression by binding the CArG motifs in their promoters. Unlike MdSOC1, MdAGL24-like might indirectly promote the expression of MdLFY by upregulating the expression of MdSOC1. Ectopic expression of MdAGL24-like in wild-type Arabidopsis induced early flowering like the phenotypes induced by other AGL24 genes. Similar to AGL24 in Arabidopsis, MdAGL24-like could rescue the late-flowering phenotype of the agl24 mutant to some extent. These results help clarify the molecular mechanism underlying flowering and provide a means of shortening the juvenile period in red-fleshed apples and other fruit trees., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

In plants, flavonoids play critical roles in resistance to biotic and abiotic stresses, and contribute substantially to the quality, flavor, and nutritional quality of many fruit crops. In apple (Malus × domestica), inbreeding has resulted in severe decreases in the genetic diversity and flavonoid content. Over the last decade, we have focused on the genetic improvement of apple using wild red-fleshed apple resources (Malus sieversii f. niedzwetzkyana). Here, we found that the MYB transcription factors (TFs) involved in the synthesis of proanthocyanidins can be classified into TT2 and PA1 types. We characterized a PA1-type MYB transcription factor, MdMYBPA1, from red-fleshed apple and identified its role in flavonoid biosynthesis using overexpression and knockdown-expression transgenes in apple calli. We explored the relationship between TT2- and PA1-type MYB TFs, and found that MdMYB9/11/12 bind the MdMYBPA1 promoter. In addition, MdMYBPA1 responded to low temperature by redirecting the flavonoid biosynthetic pathway from proanthocyanidin to anthocyanin production. In binding analyses, MdbHLH33 directly bound to the low-temperature-responsive (LTR) cis-element of the MdMYBPA1 promoter and promotes its activity. In addition, the calli expressing both MdMYBPA1 and MdbHLH33, which together form a complex, produced more anthocyanin under low temperature. Our findings shed light on the essential roles of PA1-type TFs in the metabolic network of proanthocyanidin and anthocyanin synthesis in plants. Studies on red-fleshed wild apple are beneficial for breeding new apple varieties with high flavonoid contents., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)