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

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

Tree Physiology ◽

10.1093/treephys/tpaa004 ◽

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.

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The Purple Leaf (pl6) Mutation Regulates Leaf Color by Altering the Anthocyanin and Chlorophyll Contents in Rice

Plants ◽

10.3390/plants9111477 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1477

Author(s):

Asadullah Khan ◽

Sanaullah Jalil ◽

Huan Cao ◽

Yohannes Tsago ◽

Mustapha Sunusi ◽

...

Keyword(s):

Gamma Ray ◽

Anthocyanin Biosynthesis ◽

Light Attenuation ◽

Transcript Level ◽

Underlying Mechanism ◽

Anthocyanin Accumulation ◽

Morphological Marker ◽

Chlorophyll Contents ◽

Rice Mutant ◽

Purple Coloration

The anthocyanin biosynthesis attracts strong interest due to the potential antioxidant value and as an important morphological marker. However, the underlying mechanism of anthocyanin accumulation in plant tissues is not clearly understood. Here, a rice mutant with a purple color in the leaf blade, named pl6, was developed from wild type (WT), Zhenong 41, with gamma ray treatment. By map-based cloning, the OsPL6 gene was located on the short arm of chromosome 6. The multiple mutations, such as single nucleotide polymorphism (SNP) at −702, −598, −450, an insertion at −119 in the promoter, three SNPs and one 6-bp deletion in the 5′-UTR region, were identified, which could upregulate the expression of OsPL6 to accumulate anthocyanin. Subsequently, the transcript level of structural genes in the anthocyanin biosynthesis pathway, including OsCHS, OsPAL, OsF3H and OsF3′H, was elevated significantly. Histological analysis revealed that the light attenuation feature of anthocyanin has degraded the grana and stroma thylakoids, which resulted in poor photosynthetic efficiency of purple leaves. Despite this, the photoabatement and antioxidative activity of anthocyanin have better equipped the pl6 mutant to minimize the oxidative damage. Moreover, the contents of abscisic acid (ABA) and cytokanin (CK) were elevated along with anthocyanin accumulation in the pl6 mutant. In conclusion, our results demonstrate that activation of OsPL6 could be responsible for the purple coloration in leaves by accumulating excessive anthocyanin and further reveal that anthocyanin acts as a strong antioxidant to scavenge reactive oxygen species (ROS) and thus play an important role in tissue maintenance.

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Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1703718114 ◽

2017 ◽

Vol 114 (31) ◽

pp. 8283-8288 ◽

Cited By ~ 24

Author(s):

Jun Liu ◽

Yong Juan Zhao ◽

Wan Hua Li ◽

Yun Nan Hou ◽

Ting Li ◽

...

Keyword(s):

Catalytic Domain ◽

Bimolecular Fluorescence Complementation ◽

Knock Out ◽

Guide Rna ◽

Type Iii ◽

Cyclic Adp Ribose ◽

Human Multiple Myeloma ◽

Hybrid Screening

CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.

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

Frontiers in Plant Science ◽

10.3389/fpls.2021.757936 ◽

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.

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

Horticulture Research ◽

10.1038/s41438-020-00457-z ◽

2021 ◽

Vol 8 (1) ◽

Cited By ~ 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.

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Subcellular Localization of Seed-Expressed LEA_4 Proteins Reveals Liquid-Liquid Phase Separation for LEA9 and for LEA48 Homo- and LEA42-LEA48 Heterodimers

Biomolecules ◽

10.3390/biom11121770 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1770

Author(s):

Orarat Ginsawaeng ◽

Carolin Heise ◽

Rohit Sangwan ◽

Daniel Karcher ◽

Itzell Euridice Hernández-Sánchez ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Subcellular Localization ◽

Fluorescent Protein ◽

Plant Stress ◽

Liquid Phase Separation ◽

Bimolecular Fluorescence Complementation ◽

Bioinformatic Tools ◽

Liquid Liquid Phase Separation

LEA proteins are involved in plant stress tolerance. In Arabidopsis, the LEA_4 Pfam group is the biggest group with the majority of its members being expressed in dry seeds. To assess subcellular localization in vivo, we investigated 11 seed-expressed LEA_4 proteins in embryos dissected from dry seeds expressing LEA_4 fusion proteins under its native promoters with the Venus fluorescent protein (proLEA_4::LEA_4:Venus). LEA_4 proteins were shown to be localized in the endoplasmic reticulum, nucleus, mitochondria, and plastids. LEA9, in addition to the nucleus, was also found in cytoplasmic condensates in dry seeds dependent on cellular hydration level. Most investigated LEA_4 proteins were detected in 4-d-old seedlings. In addition, we assessed bioinformatic tools for predicting subcellular localization and promoter motifs of 11 seed-expressed LEA_4 proteins. Ratiometric bimolecular fluorescence complementation assays showed that LEA7, LEA29, and LEA48 form homodimers while heterodimers were formed between LEA7-LEA29 and LEA42-LEA48 in tobacco leaves. Interestingly, LEA48 homodimers and LEA42-LEA48 heterodimers formed droplets structures with liquid-like behavior. These structures, along with LEA9 cytoplasmic condensates, may have been formed through liquid-liquid phase separation. These findings suggest possible important roles of LLPS for LEA protein functions.

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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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Transcription Factor Runx1 Activates Opn to Promote Tumor Progression in Head and Neck Cancer

10.21203/rs.3.rs-34679/v1 ◽

2020 ◽

Author(s):

Kai Liu ◽

Huiying Hu ◽

Huanyu Jiang ◽

Haidong Zhang ◽

Shanchun Gong ◽

...

Keyword(s):

Transcription Factor ◽

Head And Neck ◽

Therapeutic Potential ◽

Underlying Mechanism ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Metastatic Progression ◽

Related Transcription Factor

Abstract Background:Metastatic progression remains a major burden for head and neck squamous cell carcinoma (HNSCC). Runt-related transcription factor 1 (RUNX1)has been reported to be associated with an aggressive phenotype in several cancers. However, the precise roles of RUNX1 underlying the metastaticprogression of HNSCC remain largely unknown.Methods:RUNX1 expression levels in HNSCC cells and tissues were detected by quantitative real-time PCR (qPCR), Western blottingand immunohistochemistry (IHC). In vitro and in vivo assays were performed to investigate the function of RUNX1 in the metastatic phenotype and the tumorigenic capability of HNSCC cells. Luciferase reporter and chromatin immunoprecipitation (ChIP)-qPCR assays were performed to determine the underlying mechanism of RUNX1-mediated HNSCC aggressiveness.Results:RUNX1 was increased with disease progression in patients withHNSCC.Furthermore, we found that silencing ofRUNX1 significantly decelerated the malignant progression of HNSCC cells and reduced Osteopontin (OPN) expression in vitro, and weakened the tumorigenicityof HNSCC cells in vivo. Mechanistically, we demonstrated that RUNX1 played an important role in activating MAPK signaling by directly binding to the promoter of OPN.Conclusions: Our results provide new insight into the mechanisms underlying the facilitate metastasisability of RUNX1and reveal the therapeutic potential of targeting RUNX1 in HNSCC.

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