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

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.

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MdMYB3 helps regulate anthocyanin accumulation in apple calli under moderately acidic conditions

10.1101/429456 ◽

2018 ◽

Author(s):

Yi-Cheng Wang ◽

Jing-Jing Sun ◽

Yan-Fen Qiu ◽

Xiao-Jun Gong ◽

Li Ma ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Myb Transcription Factor ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Mobility Shift ◽

In The Wild ◽

Acidic Conditions ◽

Electrophoretic Mobility Shift Assays

AbstractAnthocyanins are the key factors controlling the coloration of plant tissues. However, the molecular mechanism underlying the effects of environmental pH on the synthesis of apple anthocyanins is unclear. In this study, we analyzed the anthocyanin contents of apple calli cultured in media at different pHs (5.5, 6.0, and 6.5). The highest anthocyanin content was observed at pH 6.0. Additionally, the moderately acidic conditions up-regulated the expression of MdMYB3 as well as specific anthocyanin biosynthesis structural genes (MdDFR and MdUFGT). Moreover, the anthocyanin content was higher in calli overexpressing MdMYB3 than in the wild-type controls at different pHs. Yeast one-hybrid assay results indicated that MdMYB3 binds to the MdDFR and MdUFGT promoters in vivo. An analysis of the MdDFR and MdUFGT promoters revealed multiple MYB-binding sites. Meanwhile, electrophoretic mobility shift assays confirmed that MdMYB3 binds to the MdDFR and MdUFGT promoters in vitro. Furthermore, GUS promoter activity assays suggested that the MdDFR and MdUFGT promoter activities are enhanced by acidic conditions, and the binding of MdMYB3 may further enhance activity. These results implied that an acid-induced apple MYB transcription factor (MdMYB3) promotes anthocyanin accumulation by up-regulating the expression of MdDFR and MdUFGT under moderately acidic conditions.

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

Horticulture Research ◽

10.1038/s41438-021-00595-y ◽

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.

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

Plants ◽

10.3390/plants10112466 ◽

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.

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

Frontiers in Plant Science ◽

10.3389/fpls.2021.774446 ◽

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.

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Anthocyanin Accumulation and Related Gene Expression Profile in ‘Red Zaosu’ Pear and Its Green Mutant

Agriculture ◽

10.3390/agriculture11090898 ◽

2021 ◽

Vol 11 (9) ◽

pp. 898

Author(s):

Yunting Zhang ◽

Shanlin Li ◽

Xianjie Gu ◽

Diya Lei ◽

Bing Zhao ◽

...

Keyword(s):

Gene Expression ◽

Anthocyanin Biosynthesis ◽

Expression Profiles ◽

Expression Patterns ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Biosynthetic Genes ◽

Specific Expression ◽

Anthocyanin Biosynthetic Genes ◽

Green Mutant

Red-skinned pear is a promising commercial fruit due to its attractive appearance and nutritious value. Anthocyanin is the determinant of the red coloration of the pear peel. However, differences in anthocyanin accumulation exist among red pear cultivars with different genetic backgrounds. In this study, we analyzed the anthocyanin content and gene expression patterns in the fruits and different tissues of the red pear ‘Red Zaosu’ at different developmental stages and found a difference in anthocyanin accumulation between ‘Red Zaosu’ pear and its green mutant. The data showed that the expression profiles of transcripts that encoded critical anthocyanin biosynthetic genes were basically consistent with a tendency to a decreased anthocyanin content during fruit development, indicating that a synergistic effect of these genes was responsible for anthocyanin biosynthesis and regulation. Tissue-specific expression analysis of anthocyanin biosynthetic genes showed that they could be expressed in all tissues but at different levels. PbF3H, PbDFR, and PbANS were mainly expressed during the early flowering period, which explained the reduced levels of anthocyanin content in petals. Additionally, the content of anthocyanins and the expression levels of PbDFR, PbANS, and PbMYB10 significantly decreased in the green mutant of ‘Red Zaosu’, suggesting that PbDFR, PbANS, and PbMYB10 probably play a decisive role in determining the skin coloration of ‘Red Zaosu’ and its green mutant.

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Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

International Journal of Molecular Sciences ◽

10.3390/ijms20153679 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3679 ◽

Cited By ~ 3

Author(s):

Lin Chen ◽

Alyne Simões ◽

Zujian Chen ◽

Yan Zhao ◽

Xinming Wu ◽

...

Keyword(s):

Wound Healing ◽

Oral Mucosa ◽

Wound Closure ◽

Expression Patterns ◽

Skin Wound ◽

Skin Wound Healing ◽

Specific Expression ◽

Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

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RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽

10.1038/s41388-021-01877-4 ◽

2021 ◽

Author(s):

Jiuna Zhang ◽

Xiaoyu Jiang ◽

Jie Yin ◽

Shiying Dou ◽

Xiaoli Xie ◽

...

Keyword(s):

Colorectal Cancer ◽

Plasma Membrane ◽

Tumor Growth ◽

Cancer Progression ◽

Critical Role ◽

Ring Finger ◽

Immunohistochemical Analysis ◽

Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

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Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽

10.3390/plants10040776 ◽

2021 ◽

Vol 10 (4) ◽

pp. 776

Author(s):

Shipra Kumari ◽

Bashistha Kumar Kanth ◽

Ju young Ahn ◽

Jong Hwa Kim ◽

Geung-Joo Lee

Keyword(s):

Abiotic Stress ◽

Biotic Stress ◽

Developmental Stages ◽

Expression Patterns ◽

Lilium Longiflorum ◽

Biotic And Abiotic Stress ◽

Biotic Stresses ◽

Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

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Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

Molecular Cancer ◽

10.1186/s12943-021-01314-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Junjie Cen ◽

Yanping Liang ◽

Yong Huang ◽

Yihui Pan ◽

Guannan Shu ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Target Genes ◽

Expression Patterns ◽

Luciferase Reporter ◽

Circular Rnas ◽

Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

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The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in ”Red Zaosu” (PyrusBretschneideri Rehd.) Pear Fruit

International Journal of Molecular Sciences ◽

10.3390/ijms22063026 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3026

Author(s):

Xieyu Li ◽

Fangxin Xiang ◽

Wei Han ◽

Bingqing Qie ◽

Rui Zhai ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Anthocyanin Biosynthesis ◽

Bimolecular Fluorescence Complementation ◽

Anthocyanin Content ◽

Pear Fruit ◽

Fruit Peel ◽

Interaction Domain ◽

Dihydroflavonol Reductase ◽

Anthocyanin Biosynthetic Pathway ◽

R2r3 Myb

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in ”Zaosu” pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

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