scholarly journals The RNA Directed DNA Methylation (RdDM) Pathway Regulates Anthocyanin Biosynthesis in Crabapple (Malus cv. spp.) Leaves by Methylating the McCOP1 Promoter

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2466
Author(s):  
Yifan Xing ◽  
Ziyi Xie ◽  
Weilei Sun ◽  
Yuying Sun ◽  
Zhenyun Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Level ◽  
Bisulfite Sequencing ◽  
Anthocyanin Biosynthesis ◽  
Apple Fruit ◽  
Bimolecular Fluorescence Complementation ◽  
Rna Expression ◽  
Anthocyanin Accumulation ◽  
Transcript Levels ◽  
Direct Target

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.

A novel NAC transcription factor, MdNAC42, regulates anthocyanin accumulation in red-fleshed apple by interacting with MdMYB10

2020 ◽  
Vol 40 (3) ◽  
pp. 413-423
Author(s):  
Shuangyi Zhang ◽  
Yixi Chen ◽  
Lingling Zhao ◽  
Chenqi Li ◽  
Jingyun Yu ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Expression Patterns ◽  
Apple Fruit ◽  
Bimolecular Fluorescence Complementation ◽  
Anthocyanin Content ◽  
Anthocyanin Accumulation ◽  
Flavonoid Pathway ◽  
Anthocyanin Pigmentation

Abstract Anthocyanin pigmentation is an important consumption trait of apple (Malus domestica Borkh.). In this study, we focused on the identification of NAC (NAM, ATAF1/2 and CUC2) proteins involved in the regulation of anthocyanin accumulation in apple flesh. A group of MdNACs was selected for comparison of expression patterns between the white-fleshed cultivar ‘Granny Smith’ and red-fleshed ‘Redlove’. Among them, MdNAC42 was screened, which exhibited a higher expression level in red-fleshed than in white-fleshed fruit, and has a positive correlation with anthocyanin content as fruits ripened. Moreover, overexpression of MdNAC42 in apple calli resulted in the up-regulation of flavonoid pathway genes, including MdCHS, MdCHI, MdF3H, MdDFR, MdANS and MdUFGT, thereby increasing the accumulation of anthocyanins, which confirmed the roles of MdNAC42 in anthocyanin biosynthesis. Notably, MdNAC42 was demonstrated to have an obvious interaction with MdMYB10 either in vitro or in vivo by yeast two-hybrid combined with bimolecular fluorescence complementation, further suggesting that MdNAC42 is an important part of the regulatory network controlling the anthocyanin pigmentation of red-fleshed apples. To the best of our knowledge, this is the first report identifying the MdNAC gene as related to anthocyanin accumulation in red-fleshed apples. This study provides valuable information for improving the regulatory model of anthocyanin biosynthesis in apple fruit.

scholarly journals MdBBX21, a B-Box Protein, Positively Regulates Light-Induced Anthocyanin Accumulation in Apple Peel

2021 ◽  
Vol 12 ◽  
Author(s):  
Bo Zhang ◽  
Zhen-Zhen Zhu ◽  
Dong Qu ◽  
Bo-Chen Wang ◽  
Ni-Ni Hao ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Apple Fruit ◽  
Bimolecular Fluorescence Complementation ◽  
Anthocyanin Accumulation ◽  
Rna Seq ◽  
Yeast Two Hybrid ◽  
Fruit Peel ◽  
Apple Peel ◽  
Its Gene ◽  
Two Hybrid

The red coloration of apple (Malus × domestica Borkh.) is due to the accumulation of anthocyanins in the fruit peel. Light is essential for anthocyanin biosynthesis in apple. In this study, we performed a transcriptome sequencing (RNA-seq) analysis of apple fruit exposed to light after unbagging. The identified differentially expressed genes included MdBBX21, which is homologous to Arabidopsis BBX21, suggesting it may be involved in light-induced anthocyanin biosynthesis. Additionally, MdBBX21 was localized in the nucleus and its gene was expressed earlier than MdMYB1 in apple peel treated with light. Overexpressing MdBBX21 in Arabidopsis and apple calli under light increased anthocyanin accumulation. Dual-luciferase and yeast one-hybrid assays confirmed that MdBBX21 binds to the MdHY5, MdBBX20, and MdBBX22-1/2 promoters and induces expression. At the same time, MdHY5 can also activate the expression of MdBBX21. Furthermore, bimolecular fluorescence complementation and yeast two-hybrid assays demonstrated that MdBBX21 can interact with MdHY5. This interaction can significantly enhance MdMYB1 promoter activity. These findings clarify the molecular mechanism by which MdBBX21 positively regulates light-induced anthocyanin accumulation in apple.

MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple

2020 ◽  
Author(s):  
Wen-Fang Li ◽  
Gai-Xing Ning ◽  
Cun-Wu Zuo ◽  
Ming-Yu Chu ◽  
Shi-Jin Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Promoter Region ◽  
Anthocyanin Biosynthesis ◽  
Apple Fruit ◽  
Anthocyanin Synthesis ◽  
Mrna Levels ◽  
Anthocyanin Accumulation ◽  
Dna Hypomethylation ◽  
Fruit Skin

Abstract Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. ‘Red Delicious’ (G0) and its four continuous-generation bud sport mutants including ‘Starking Red’ (G1), ‘Starkrimson’ (G2), ‘Campbell Redchief’ (G3) and ‘Vallee spur’ (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin colour traits remain elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in ‘Golden Delicious’ fruits, ‘Gala’ calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in ‘Red Fuji’ apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.

scholarly journals SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2 to regulate anthocyanin biosynthesis by activating SlDFR expression in tomato

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Dan Luo ◽  
Cheng Xiong ◽  
Aihua Lin ◽  
Chunli Zhang ◽  
Wenhui Sun ◽  
...  
Keyword(s):  
Growth Regulation ◽  
Anthocyanin Biosynthesis ◽  
Plant Stress ◽  
Underlying Mechanism ◽  
Bimolecular Fluorescence Complementation ◽  
Cop9 Signalosome ◽  
Anthocyanin Accumulation ◽  
Related Transcription Factor ◽  
Hybrid Screening

AbstractAnthocyanins play vital roles in plant stress tolerance and growth regulation. Previously, we reported that the photomorphogenesis-related transcription factor SlBBX20 regulates anthocyanin accumulation in tomato. However, the underlying mechanism remains unclear. Here, we showed that SlBBX20 promotes anthocyanin biosynthesis by binding the promoter of the anthocyanin biosynthesis gene SlDFR, suggesting that SlBBX20 directly activates anthocyanin biosynthesis genes. Furthermore, we found by yeast two-hybrid screening that SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2, and the interaction was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. SlCSN5 gene silencing led to anthocyanin hyperaccumulation in the transgenic tomato calli and shoots, and SlCSN5-2 overexpression decreased anthocyanin accumulation, suggesting thSlCSN5-2 enhanced the ubiquitination of SlBBX20 and promoted the degradation of SlBBX20 in vivo. Consistently, silencing the SlCSN5-2 homolog in tobacco significantly increased the accumulation of the SlBBX20 protein. Since SlBBX20 is a vital regulator of photomorphogenesis, the SlBBX20-SlCSN5-2 module may represent a novel regulatory pathway in light-induced anthocyanin biosynthesis.

scholarly journals The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhaofei Duan ◽  
Shiyu Tian ◽  
Guobin Yang ◽  
Min Wei ◽  
Jing Li ◽  
...  
Keyword(s):  
Plant Species ◽  
Anthocyanin Biosynthesis ◽  
Solanum Melongena ◽  
Amino Acid Sequences ◽  
Negative Regulator ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Assay ◽  
Anthocyanin Accumulation ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

Many basic helix-loop-helix transcription factors (TFs) have been reported to promote anthocyanin biosynthesis in numerous plant species, but little is known about bHLH TFs that inhibit anthocyanin accumulation. In this study, SmbHLH1 from Solanum melongena was identified as a negative regulator of anthocyanin biosynthesis. However, SmbHLH1 showed high identity with SmTT8, which acts as a SmMYB113-dependent positive regulator of anthocyanin-biosynthesis in plants. Overexpression of SmbHLH1 in eggplant caused a dramatic decrease in anthocyanin accumulation. Only the amino acid sequences at the N and C termini of SmbHLH1 differed from the SmTT8 sequence. Expression analysis revealed that the expression pattern of SmbHLH1 was opposite to that of anthocyanin accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SmbHLH1 could not interact with SmMYB113. Dual-luciferase assay demonstrated that SmbHLH1 directly repressed the expression of SmDFR and SmANS. Our results demonstrate that the biological function of bHLHs in anthocyanin biosynthesis may have evolved and provide new insight into the molecular functions of orthologous genes from different plant species.

scholarly journals Coordinated Regulation of Biosynthetic and Regulatory Genes Coincides with Anthocyanin Accumulation in Developing Eggplant Fruit

2015 ◽  
Vol 140 (2) ◽  
pp. 129-135 ◽  
Author(s):  
John R. Stommel ◽  
Judith M. Dumm
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Molecular Genetic ◽  
Solanum Melongena ◽  
Regulatory Genes ◽  
Model Systems ◽  
Anthocyanin Content ◽  
Anthocyanin Accumulation ◽  
Transcript Levels ◽  
Dark Violet ◽  
Marketable Fruit

Violet to black pigmentation of eggplant (Solanum melongena L.) fruit is caused by anthocyanin accumulation. Model systems demonstrate the role of regulatory genes in the control of anthocyanin biosynthesis. Anthocyanin structural gene transcription requires the expression of at least one member of each of three transcription factor families: MYB, MYC, and WD. To determine the molecular genetic basis for anthocyanin pigmentation in eggplant fruit, we used real-time polymerase chain reaction (PCR) to evaluate the expression of anthocyanin biosynthetic (Chs, Dfr, Ans) and regulatory (Myc, MybB, MybC, Wd) genes in S. melongena genotypes that produce fruit with dark violet (‘Classic’) or white (‘Ghostbuster’) coloration, respectively. Transcript levels and anthocyanin content were evaluated in fruit at various stages of development ranging from small post-anthesis fruit to full-sized marketable fruit. Anthocyanin content increased 9-fold in developing violet-colored ‘Classic’ fruit, whereas low but detectable concentrations were found in white ‘Ghostbuster’ fruit. Chs, Dfr, and Ans as well as MybC and Myc transcript levels were significantly higher in ‘Classic’ in comparison with ‘Ghostbuster’ fruit at comparable stages of fruit development with greatest differences observed for Ans transcript levels. MybC and Myc transcript levels increased in developing ‘Classic’ fruit coincident with increasing anthocyanin content. MybB and Wd transcript levels were not coordinated with changes in biosynthetic transcript levels or anthocyanin concentration.

scholarly journals The Structure and Methylation Level of the McMYB10 Promoter Determine the Leaf Color of Malus Crabapple

HortScience ◽  
2017 ◽  
Vol 52 (4) ◽  
pp. 520-526 ◽  
Author(s):  
Ji Tian ◽  
Ke-ting Li ◽  
Shi-ya Zhang ◽  
Jie Zhang ◽  
Ting-ting Song ◽  
...  
Keyword(s):  
Methylation Level ◽  
Anthocyanin Biosynthesis ◽  
Anthocyanin Accumulation ◽  
Substantial Variation ◽  
Leaf Color ◽  
Human Diet ◽  
Development Stages ◽  
Leaf Coloration ◽  
The Relationship ◽  
Myb10 Transcription Factor

Anthocyanins are protective pigments that accumulate in plant organs such as fruits and leaves, and are nutritionally valuable components of the human diet. The MYB10 transcription factor (TF) plays an important role in regulating anthocyanin biosynthesis in Malus crabapple leaves. However, little is known about how the promoter regulates McMYB10 expression and influences the substantial variation in leaf anthocyanin accumulation and coloration that is observed in different crabapple cultivars. In this study, we analyzed leaf coloration, anthocyanin levels, and the expression levels of McMYB10 in the leaves of 15 crabapple cultivars with three leaf colors at various development stages, and showed that the expression of McMYB10 correlates positively with anthocyanin accumulation. We also examined the relationship between the number of R6 and R1 elements in the McMYB10 promoters of the different cultivars and the pigmentation of the new buds of spring-red cultivars, as well as the methylation level of the McMYB10 promoters at different development stages in three representative crabapple cultivars. The ratio of R6/R1 minisatellites in the promoters correlated with the color and anthocyanin accumulation in new crabapple buds, and we concluded that the differences in promoter structure and methylation level of the McMYB10 promoters coordinately affect the leaf color of crabapple cultivars.

scholarly journals The apple 14-3-3 protein MdGRF11 interacts with the BTB protein MdBT2 to regulate nitrate deficiency-induced anthocyanin accumulation

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Yi-Ran Ren ◽  
Qiang Zhao ◽  
Yu-Ying Yang ◽  
Tian-En Zhang ◽  
Xiao-Fei Wang ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Anthocyanin Biosynthesis ◽  
Critical Role ◽  
26S Proteasome ◽  
Bimolecular Fluorescence Complementation ◽  
Anthocyanin Accumulation ◽  
The Stability ◽  
Dependent Pathway ◽  
Two Hybrid

AbstractNitrogen is an important factor that affects plant anthocyanin accumulation. In apple, the nitrate-responsive BTB/TAZ protein MdBT2 negatively regulates anthocyanin biosynthesis. In this study, we found that MdBT2 undergoes posttranslational modifications in response to nitrate deficiency. Yeast two-hybrid, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with MdGRF11, a 14-3-3 protein; 14-3-3 proteins compose a family of highly conserved phosphopeptide-binding proteins involved in multiple physiological and biological processes. The interaction of MdGRF11 negatively regulated the stability of the MdBT2 protein via a 26S proteasome-dependent pathway, which increased the abundance of MdMYB1 proteins to activate the expression of anthocyanin biosynthesis-related genes. Taken together, the results demonstrate the critical role of 14-3-3 proteins in the regulation of nitrate deficiency-induced anthocyanin accumulation. Our results provide a novel avenue to elucidate the mechanism underlying the induction of anthocyanin biosynthesis in response to nitrate deficiency.

Whole-genome DNA methylation patterns and complex associations with gene expression associated with anthocyanin biosynthesis in apple fruit skin

Planta ◽  
2019 ◽  
Vol 250 (6) ◽  
pp. 1833-1847 ◽  
Author(s):  
Wen-Fang Li ◽  
Gai-Xing Ning ◽  
Juan Mao ◽  
Zhi-Gang Guo ◽  
Qi Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Anthocyanin Biosynthesis ◽  
Apple Fruit ◽  
Whole Genome ◽  
Fruit Skin ◽  
Methylation Patterns

scholarly journals Transposon-induced methylation of the RsMYB1 promoter disturbs anthocyanin accumulation in red-fleshed radish

2020 ◽  
Vol 71 (9) ◽  
pp. 2537-2550 ◽  
Author(s):  
Qingbiao Wang ◽  
Yanping Wang ◽  
Honghe Sun ◽  
Liang Sun ◽  
Li Zhang
Keyword(s):  
Dna Methylation ◽  
Raphanus Sativus ◽  
Promoter Region ◽  
Sequence Variation ◽  
Anthocyanin Biosynthesis ◽  
Underlying Mechanism ◽  
Anthocyanin Accumulation ◽  
Virus Induced Gene Silencing ◽  
Genome Resequencing ◽  
Raphanus Sativus L

Abstract Red-fleshed radish (Raphanus sativus L.) is a unique cultivar whose taproot is rich in anthocyanins beneficial to human health. However, the frequent occurrence of white-fleshed mutants affects the purity of commercially produced radish and the underlying mechanism has puzzled breeders for many years. In this study, we combined quantitative trait location by genome resequencing and transcriptome analyses to identify a candidate gene (RsMYB1) responsible for anthocyanin accumulation in red-fleshed radish. However, no sequence variation was found in the coding and regulatory regions of the RsMYB1 genes of red-fleshed (MTH01) and white-fleshed (JC01) lines, and a 7372 bp CACTA transposon in the RsMYB1 promoter region occurred in both lines. A subsequent analysis suggested that the white-fleshed mutant was the result of altered DNA methylation in the RsMYB1 promoter. This heritable epigenetic change was due to the hypermethylated CACTA transposon, which induced the spreading of DNA methylation to the promoter region of RsMYB1. Thus, RsMYB1 expression was considerably down-regulated, which inhibited anthocyanin biosynthesis in the white-fleshed mutant. An examination of transgenic radish calli and the results of a virus-induced gene silencing experiment confirmed that RsMYB1 is responsible for anthocyanin accumulation. Moreover, the mutant phenotype was partially eliminated by treatment with a demethylating agent. This study explains the molecular mechanism regulating the appearance of white-fleshed mutants of red-fleshed radish.

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