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

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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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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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 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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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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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 role of Clt1-regulated-Xylan metabolism in the melanin and toxin formation for the pathogenicity of Curvularia lunata in Maize

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-20-0235-r ◽

2021 ◽

Author(s):

Jinxin Gao ◽

Jie Chen

Keyword(s):

Toxin Production ◽

Bimolecular Fluorescence Complementation ◽

Curvularia Lunata ◽

Yeast Two Hybrid ◽

Acetyl Xylan Esterase ◽

Btb Domain ◽

Intermediate Metabolites ◽

Regulative Mechanism ◽

Two Hybrid

We previously reported that the BTB domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata were regulated by Clt1. Yeast two-hybrid (Y2H), and bimolecular ﬂuorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin, as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.

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Nighttime Temperatures and Sunlight Intensities Interact to Influence Anthocyanin Biosynthesis and Photooxidative Sunburn in “Fuji” Apple

Frontiers in Plant Science ◽

10.3389/fpls.2021.694954 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiaomin Xue ◽

Ying Duan ◽

Jinzheng Wang ◽

Fengwang Ma ◽

Pengmin Li

Keyword(s):

Light Intensity ◽

Low Temperatures ◽

Anthocyanin Biosynthesis ◽

High Light ◽

Anthocyanin Accumulation ◽

Low Light ◽

Low Light Intensity ◽

Apple Peel ◽

Fuji Apple ◽

Nighttime Temperatures

Light and low temperatures induce anthocyanin accumulation, but intense sunlight causes photooxidative sunburn. Nonetheless, there have been few studies of anthocyanin synthesis under different sunlight intensities and low nighttime temperatures. Here, low nighttime temperatures followed by low light intensity were associated with greater anthocyanin accumulation and the expression of anthocyanin biosynthesis genes in “Fuji” apple peel. UDP-glucose flavonoid-3-O-glucosyltransferase (UFGT) activity was positively associated with anthocyanin enrichment. Ascorbic acid can be used as an electron donor of APX to scavenge H2O2 in plants, which makes it play an important role in oxidative defense. Exogenous ascorbate altered the anthocyanin accumulation and reduced the occurrence of high light–induced photooxidative sunburn by removing hydrogen peroxide from the peel. Overall, low light intensity was beneficial for the accumulation of anthocyanin and did not cause photooxidative sunburn, whereas natural light had the opposite effect on the apple peel at low nighttime temperatures. This study provides an insight into the mechanisms by which low temperatures induce apple coloration and high light intensity causes photooxidative sunburn.

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Identification of Beet necrotic yellow vein virus P25 Pathogenicity Factor–Interacting Sugar Beet Proteins That Represent Putative Virus Targets or Components of Plant Resistance

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-8-0999 ◽

2009 ◽

Vol 22 (8) ◽

pp. 999-1010 ◽

Cited By ~ 15

Author(s):

Heike Thiel ◽

Mark Varrelmann

Keyword(s):

Sugar Beet ◽

Resistance Mechanism ◽

Yellow Vein ◽

Bimolecular Fluorescence Complementation ◽

Plant Proteins ◽

Yeast Two Hybrid ◽

Response To Stress ◽

Bimolecular Fluorescence Complementation Assay ◽

Two Hybrid ◽

Important Disease

Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.

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Silencing Suppressor Protein VPg of a Potyvirus Interacts With the Plant Silencing-Related Protein SGS3

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-14-0109-r ◽

2014 ◽

Vol 27 (11) ◽

pp. 1199-1210 ◽

Cited By ~ 26

Author(s):

Minna-Liisa Rajamäki ◽

Janne Streng ◽

Jari P. T. Valkonen

Keyword(s):

Rna Silencing ◽

Bimolecular Fluorescence Complementation ◽

Mrna Levels ◽

Yeast Two Hybrid ◽

Main Determinant ◽

Potato Virus A ◽

Cytoplasmic Bodies ◽

Multiple Protein ◽

Suppressor Of Rna Silencing ◽

Two Hybrid

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg–SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg–SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.

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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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