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SUMMARY: Anthocyanin generation in apples (Malus domestica) and the pigmentation that results from it may be caused by irradiation and through administration of methyl jasmonate (MeJA). However, their regulatory interrelationships associated with fruit coloration are not well defined. To determine whether MdERF109, a transcription factor (TF) involved in light‐mediated coloration and anthocyanin biosynthesis, has synergistic effects with other proteins, we performed a yeast two‐hybrid assessment and identified another TF, MdWER. MdWER was induced by MeJA treatment, and although overexpression of MdWER alone did not promote anthocyanin accumulation co‐overexpression with MdERF109 resulted in significantly increase in anthocyanin biosynthesis. MdWER may form a protein complex with MdERF109 to promote anthocyanin accumulation by enhancing combinations between the proteins and their corresponding genes. In addition, MdWER, as a MeJA responsive protein, interacts with the anthocyanin repressor MdJAZ2. Transient co‐expression in apple fruit and protein interaction assays allowed us to conclude that MdERF109 and MdJAZ2 interact with MdWER and take part in the production of anthocyanins upon MeJA treatment and irradiation. Our findings validate a role for the MdERF109‐MdWER‐MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit. Significance Statement: Our findings validate a role for the MdERF109‐MdWER‐MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit. [ABSTRACT FROM AUTHOR]

Summary: Light induces anthocyanin accumulation and hence decides the coloration of apple fruit. It also plays a key role in regulating the biosynthesis of other secondary metabolites. However, the crosstalk between anthocyanin and lignin metabolism during light induction, which affects the edible quality and visual quality of apple fruit, respectively, have rarely been characterized.In this study, we identified and functionally elucidated the roles of miR7125 and its target, cinnamoyl‐coenzyme A reductase gene (CCR), in regulating the homeostasis between anthocyanin and lignin biosynthesis during light induction. Overexpressing miR7125 or inhibiting CCR transiently in apple fruit promoted anthocyanin biosynthesis but reduced lignin production under light‐induced conditions. Consistently, opposite results were observed under the background of repressed miR7125 or overexpressed CCR.We found that the repressor MdMYB16 and the activator MdMYB1 bound to the miR7125 promoter. Transient repression of MdMYB16 upregulated miR7125 expression significantly, accompanied by decreased levels of MdCCR transcript, resulting in a reduction in the lignin biosynthesis and an increase in anthocyanin accumulation. However, transient overexpression of MdMYB16 produced the opposite effects to MdMYB16‐RNAi.The results reveal a novel mechanism by which the MdMYB16/MdMYB1‐miR7125‐MdCCR module collaboratively regulates homeostasis between anthocyanin and lignin biosynthesis under light induction in apple. [ABSTRACT FROM AUTHOR]

Proanthocyanidins (PAs) are a class of flavonoid compounds in plants that play many important roles in pest and disease resistance and are beneficial components of the human diet. The crabapple (Malus) provides an excellent model to study PA biosynthesis and metabolism; therefore, to gain insights into the PA regulatory network in Malus plants, we performed RNA-seq profiling of fruits of the 'Flame' cultivar at five sequential developmental stages. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that differentially expressed genes (DEGs) related to the functional category 'plant hormone signal transduction' were significantly enriched during fruit development. Further analysis showed that ethylene signal transduction pathway genes or response genes, such as ERS (ethylene response sensor), EIN3 (ETHYLENE INSENSITIVE 3) and ERFs (ethylene response factors), may play an important role in the regulatory network of PA biosynthesis. Additionally, 12 DEGs, including 10 ERFs, 1 MYB, and 1 bHLH transcription factor, associated with PA biosynthesis were identified using WGCNA. The expression patterns of these genes correlated with PA accumulation trends and transcriptome data from qRT-PCR analysis. The expression of RAP2-4 (RELATED TO APETALA 2-4) and RAV1 (related to ABI3/VP1), which belong to the ERF transcription factor family, showed the greatest correlations with PAs accumulation among the 12 identified TFs. Agrobacterium mediated-transient overexpression of the RAP2-4 led to an increase in PA abundance in crabapple leaves and apple fruits, and the opposite results were observed in RAV1 -overexpressed crabapple leaves and apple fruits. Moreover, a yeast one-hybrid assay showed that RAP2-4 and RAV1 specifically bound the promoters of the PA biosynthetic genes McLAR1 and McANR2 , respectively. These results indicate that RAP2-4 act as an inducer and RAV1 act as a repressor of PA biosynthesis by regulating the expression of the PA biosynthetic genes McLAR1 and McANR2. Taken together, we identified two potential regulators of PA biosynthesis and provide new insights into the ethylene-PA regulatory network. [ABSTRACT FROM AUTHOR]

Flavonols are polyphenolic compounds that play important roles in plant stress resistance and development. They are also valuable components of the human diet. The Malus crabapple cultivar 'Flame' provides an excellent model for studying flavonol biosynthesis due to the high flavonol content of its fruit peel. To obtain a more detailed understanding of the flavonol regulatory network involved in fruit development, the transcriptomes of the fruit of the Malus cv. 'Flame' from five continuous developmental stages were analyzed using RNA sequencing. A flavonol-related gene module was identified through weighted gene coexpression network analysis (WGCNA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that phytohormones are involved in regulating flavonol biosynthesis during fruit development. A putative transcription factor, MdMYB8, was selected for further study through hub gene correlation network analysis and yeast one-hybrid assays. Stable overexpression or RNAi knockdown of MdMYB8 in transgenic 'Orin' apple calli resulted in a higher or lower flavonol content, respectively, suggesting that MdMYB8 is a regulator of flavonol biosynthesis. This transcriptome analysis provides valuable data for future studies of flavonol synthesis and regulation. [ABSTRACT FROM AUTHOR]

Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. To identify new regulatory genes in apple (Malus domestica) that may be involved in regulating low temperature induced anthocyanin biosynthesis, we performed RNA-seq analysis of leaves from the ‘Gala’ apple cultivar following exposure to a low temperature (16 °C). A visible red color appeared on the upper leaves and the anthocyanin content increased significantly after the low temperature treatment. Genes from the flavonoid biosynthesis pathway were significantly enriched among the differentially expressed genes, and the expression of several transcription factors was shown by WGCNA (weighted gene co-expression network analysis) to correlate with anthocyanin accumulation, including members of the MYB, MADS, WRKY, WD40, Zinc Finger and HB-ZIP families. Three MYB transcription factors (MdMYB12, MdMYB22 and MdMYB114), which had several CBF/DREB response elements in their promoters, were significantly induced by low temperature exposure and their expression also correlated highly with anthocyanin accumulation. We hypothesize that they may act as regulators of anthocyanin biosynthesis and be regulated by CBF/DREB transcription factors in apple leaves under low temperature conditions. The analyses presented here provide insights into the molecular mechanisms underlying anthocyanin accumulation during low temperature exposure. [ABSTRACT FROM AUTHOR]

Cuticular waxes of plant and organ surfaces play an important role in protecting plants from biotic and abiotic stress and extending the freshness, storage time and shelf life in the post-harvest agricultural products. WRI1, a transcription factor of AP2/SHEN families, had been found to trigger the related genes taking part in the biosynthesis of seed oil in many plants. But whether WRI1 is involved in the biosynthesis of the cuticular waxes on the Malus fruits surface has been unclear. We investigated the changes of wax composition and structure, the related genes and WRI1 expression on Malus asiatica Nakai and sieversii fruits with the low temperature treatments, found that low temperature induced the up-regulated expression of McWRI1, which promoted gene expression of McKCS, McLACs and McWAX in very-long-chain fatty acid biosynthesis pathway, resulting in the accumulation of alkanes component and alteration of wax structure on the fruit surface. Corresponding results were verified in McWRI1 silenced by VIGS, and WRI1 silenced down-regulated the related genes on two kinds of fruits, it caused the diversity alteration in content of some alkanes, fatty acid and ester component in two kinds of fruits. We further conducted Y1H assay to find that McWRI1 transcription factor activated the promoter of McKCS, McLAC and McWAX to regulate their expression. These results demonstrated that McWRI1 is involved in regulating the genes related synthesis of very long chain fatty acid on surface of apple fruits in storage process, providing a highlight for improvement of the modified atmosphere storage of apple fruits. [ABSTRACT FROM AUTHOR]

Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple. [ABSTRACT FROM AUTHOR]

Anthocyanin is a critical factor involved in coloration of plant tissues, but the mechanism how medium pH values affect anthocyanin accumulation in woody plants is unknown. We analyzed anthocyanin composition and the expression of elements encoding anthocyanin and flavonols biosynthesis underlying different medium pH values by using three different leave color type cultivars. HPLC analysis demonstrated that high medium pH values treatment induced a dramatic decrease in the concentration of cyaniding in crabapple leaves. Conversely, the high medium pH values induced up-regulation of the content of flavones and flavonols, suggesting that low pH treatment-induced anthocyanin accumulation. Quantitative real time PCR experiment showed the expression level of anthocyanidin synthase (McANS) and uridine diphosphate glucose flavonoid 3-O-glucosyltransferase (McUFGT) was up-regulated by low pH values treatment, and high medium pH value treatment up-regulate the transcription level of flavonol synthase (McFLS). Meanwhile, several MYB TFs have been suggested in the regulation of pH responses. These results strongly indicate that the low pH treatment-induced anthocyanin accumulation is mediated by the variation of mRNA transcription of the anthocyanin biosynthetic genes. [ABSTRACT FROM AUTHOR]

The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q) PCR to analyze the methylation level of the promoter of constitutively photomorphogenic 1 (McCOP1) from Malus cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the McCOP1 promoter was negatively correlated with McCOP1 RNA expression, and inhibiting DNA methylation caused decreased methylation of the McCOP1 promoter and asymmetric cytosine CHH methylation. We observed that the McCOP1 promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a McCOP1 promoter GGTTCGG site. Bimolecular fluorescence complementation (BIFC) analysis showed that RNA-directed DNA methylation (McRDM1) interacted with McAGO4 and another RdDM protein, domains rearranged methyltransferase 2 (McDRM2), to regulate the CHH methylation of the McCOP1 promoter. Detection of CHH methylation and COP1 gene expression in the Arabidopsis thalianaatago4, atdrm2 and atrdm1 mutants showed that RDM1 is the effector of the RdDM pathway. This was confirmed by silencing McRDM1 in crabapple leaves or apple fruit, which resulted in a decrease in McCOP1 CHH methylation and an increase in McCOP1 transcript levels, as well as in anthocyanin accumulation. In conclusion, these results show that the RdDM pathway is involved in regulating anthocyanin accumulation through CHH methylation of the McCOP1 promoter. [ABSTRACT FROM AUTHOR]

[Display omitted] • A crosstalk between copper homeostasis and ROS homeostasis mediated by the miR408a-BBP-LAC3/CSD1 module was revealed under light induction and copper stress in Malus plants. • The copper homeostasis fluctuation triggers ROS homeostasis variation and participates in modulating anthocyanin accumulationin under excessive copper and copper defficiency in Malus plants. The expression of miR408 is affected by copper (Cu) conditions and positively regulates anthocyanin biosynthesis in Arabidopsis. However, the underlying mechanisms by which miR408 regulates anthocyanin biosynthesis mediated by Cu homeostasis and reactive oxygen species (ROS) homeostasis remain unclear in Malus plants. Our study aims to elucidate how miR408a and its target, basic blue protein (BBP) regulate Cu homeostasis and ROS homeostasis, and anthocyanin biosynthesis in Malus plants. The roles of miR408a and its target BBP in regulating anthocyanin biosynthesis, Cu homeostasis, and ROS homeostasis were mainly identified in Malus plants. We found that the BBP protein interacted with the copper-binding proteins LAC3 (laccase) and CSD1 (Cu/Zn SOD superoxide dismutase), indicating a potential crosstalk between Cu homeostasis and ROS homeostasis might be mediated by miR408 to regulate the anthocyanin accumulation. Further studies showed that overexpressing miR408a or suppressing BBP transiently significantly increased the expression of genes related to Cu binding and Cu transport, leading to anthocyanin accumulation under light induction in apple fruit and Malus plantlets. Consistently, opposite results were obtained when repressing miR408a or overexpressing BBP. Moreover, light induction significantly increased the expression of miR408a , CSD1 , and LAC3 , but significantly reduced the BBP expression, resulting in increased Cu content and anthocyanin accumulation. Furthermore, excessive Cu significantly increased the anthocyanin accumulation, accompanied by reduced expression of miR408a and Cu transport genes, and upregulated expression of Cu binding proteins including BBP , LAC3 , and CSD1 to maintain the Cu homeostasis and ROS homeostasis in Malus plantlets. Our findings provide new insights into the mechanism by which the miR408a-BBP-LAC3/CSD1 module perceives light and Cu signals regulating Cu and ROS homeostasis, ultimately affecting anthocyanin biosynthesis in Malus plants. [ABSTRACT FROM AUTHOR]

Red leaf color is an attractive trait of Malus families, including crabapple (Malus spp.); however, little is known about the molecular mechanisms that regulate the coloration. Dihydroflavonols are intermediates in the production of both colored anthocyanins and colorless flavonols, and this current study focused on the gene expression balance involved in the relative accumulation of these compounds in crabapple leaves. Levels of anthocyanins and the transcript abundances of the anthocyanin biosynthetic gene, dihydroflavonol 4-reductase (McDFR) and the flavonol biosynthetic gene, flavonol synthase (McFLS), were assessed during the leaf development in two crabapple cultivars, 'Royalty' and 'Flame'. The concentrations of anthocyanins and flavonols correlated with leaf color and we propose that the expression of McDFR and McFLS influences their accumulation. Further studies showed that overexpression of McDFR, or silencing of McFLS, increased anthocyanin production, resulting in red-leaf and red fruit peel phenotypes. Conversely, elevated flavonol production and green phenotypes in crabapple leaves and apple peel were observed when McFLS was overexpressed or McDFR was silenced. These results suggest that the relative activities of McDFR and McFLS are important determinants of the red color of crabapple leaves, via the regulation of the metabolic fate of substrates that these enzymes have in common. [ABSTRACT FROM AUTHOR]

Fruit pigment accumulation, which represents an important indicator of nutrient quality and appearance value, is often affected by low light under rain, cloud, fog and haze conditions during the veraison period. It is not known whether continuous low light interferes with the production and accumulation of secondary metabolites in veraison fruit. In this paper, we measured pigments and the transcriptional level of genes related to secondary metabolites, i.e., flavonoid biosynthesis in the peel and flesh of Malus crabapple ‘Radiant’ fruit in response to normal light and shade from 10th July to 30th August. The results showed crosstalk between the flavonoid biosynthetic genes and the involvement of key transcription factors such as McMYB4, McMYB7, McMYB10, and McMYB16 in the regulation of the ratio of anthocyanins and flavanols, which accounted for the different colouration of the fruit peel and flesh under shade conditions. A model is proposed for the regulation of the flavonoid pathway in the peel and flesh of ‘Radiant’ fruit based on our study results. Moreover, the molecular mechanism for ‘Radiant’ fruit colouration provides reference information for understanding the light regulatory mechanism involved in the biosynthesis of flavonoids and for designing the next generation of apple breeding. [ABSTRACT FROM AUTHOR]

Highlights: [•] McMYB10 encoded an R2R3 MYB family transcription factor in Malus crabapple. [•] The transcription level of McMYB10 was consistent with the content of anthocyanin. [•] Overexpression of McMYB10 in tobacco generated highly pigmentation in the petals. [•] NtDFR and NtANS were significantly activated by McMYB10. [Copyright &y& Elsevier]