Cloning and Characterization of a Tryptophan–Aspartic Acid Repeat Gene Associated with the Regulation of Anthocyanin Biosynthesis in Oriental Hybrid Lily

Anthocyanins are major pigments responsible for the color of lily (Lilium sp.) flowers. Anthocyanin synthesis is part of the flavonoid metabolic pathway. Numerous transcription factors, including R2R3-MYBs, basic helix-loop-helix (bHLH), and tryptophan–aspartic acid repeat (also known as WD40 or WD repeat) proteins, known to regulate flavonoid biosynthesis have been identified in various plant species. However, there is limited information available on WD repeat proteins in lilies. In this study, we identified a WD repeat gene in the Oriental hybrid lily ‘Sorbonne’ (Lilium hybrid WD repeat, LhWDR). LhWDR contains no introns, and has a 1100–base pair open reading frame, encoding a putative protein of 370 amino acids. LhWDR was found to be localized in the cytoplasm of transgenic Arabidopsis thaliana root cells. Expression patterns of LhWDR in different organs and at different periods of lily tepal growth revealed that the expression levels of this gene are closely associated with anthocyanin accumulation. A yeast two-hybrid assay demonstrated that full-length LhWDR interacts with the 420 N-terminal amino acids of Lilium hybrid bHLH2. Interestingly, overexpression of LhWDR in A. thaliana led to an upregulation of the dihydroflavonol 4-reductase gene, which is an important structural gene downstream of the anthocyanin pathway. These results indicate that the WD repeat protein LhWDR might interact with a bHLH transcription factor to regulate anthocyanin biosynthesis.

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Transcriptome Analysis Revealed the Mechanism by Which Exogenous ABA Increases Anthocyanins in Blueberry Fruit During Veraison

Frontiers in Plant Science ◽

10.3389/fpls.2021.758215 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tianyu Han ◽

Wenlong Wu ◽

Weilin Li

Keyword(s):

Transcription Factors ◽

Fruit Ripening ◽

Expression Patterns ◽

Synthesis Process ◽

Mature Stage ◽

Anthocyanin Synthesis ◽

Bhlh Transcription Factor ◽

Anthocyanin Content ◽

Anthocyanin Pathway ◽

Green Stage

Blueberry (Vaccinium spp.) is a popular healthy fruit worldwide. The health value of blueberry is mainly because the fruit is rich in anthocyanins, which have a strong antioxidant capacity. However, because blueberry is a non-model plant, little is known about the structural and regulatory genes involved in anthocyanin synthesis in blueberries. Previous studies have found that spraying 1,000 mg/L abscisic acid at the late green stage of “Jersey” highbush blueberry fruits can increase the content of anthocyanins. In this experiment, the previous results were verified in “Brightwell” rabbiteye blueberry fruits. Based on the previous results, the anthocyanin accumulation process in blueberry can be divided into six stages from the late green stage to the mature stage, and the transcriptome was used to systematically analyze the blueberry anthocyanin synthesis process. Combined with data from previous studies on important transcription factors regulating anthocyanin synthesis in plants, phylogenetic trees were constructed to explore the key transcription factors during blueberry fruit ripening. The results showed that ABA increased the anthocyanin content of blueberry fruits during veraison. All structural genes and transcription factors (MYB, bHLH, and WD40) involved in the anthocyanin pathway were identified, and their spatiotemporal expression patterns were analyzed. The expression of CHS, CHI, DFR, and LDOX/ANS in ABA-treated fruits was higher in the last two stages of maturity, which was consistent with the change in the anthocyanin contents in fruits. In general, six MYB transcription factors, one bHLH transcription factor and four WD40 transcription factors were found to change significantly under treatment during fruit ripening. Among them, VcMYBA plays a major role in the regulation of anthocyanin synthesis in ABA signaling. This result preliminarily explained the mechanism by which ABA increases the anthocyanin content and improves the efficiency of the industrial use of blueberry anthocyanins.

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Transcriptome Analysis Revealed The Mechanism of Exogenous ABA Increasing Anthocyanins in Blueberry Fruit During Veraison

10.21203/rs.3.rs-88852/v1 ◽

2020 ◽

Author(s):

Tianyu Han ◽

Zhixiang Yan ◽

Wenlong Wu ◽

Weilin Li

Keyword(s):

Transcription Factors ◽

Transcriptome Analysis ◽

Expression Patterns ◽

Synthesis Process ◽

Anthocyanin Synthesis ◽

Bhlh Transcription Factor ◽

Anthocyanin Content ◽

Structural Genes ◽

Anthocyanin Pathway ◽

Green Stage

Abstract Background: Blueberry（Vaccinium Spp）is a popular healthy fruit all over the world. The health value of blueberry is mainly due to the fact that blueberry is rich in anthocyanins, which have a strong antioxidant capacity. However, due to the fact that blueberry is a non model plant, little is known about the structural genes and regulatory genes involved in the anthocyanin synthesis of blueberries. Previous studies have found that spraying abscisic acid at the late green stage of blueberry fruit can increase the content of anthocyanins. Based on the former results, the anthocyanin accumulation process of blueberry can be divided into six stages from late green stage to mature stage to analyze the anthocyanin synthesis mechanism. In order to identify the important genes in the anthocyanin synthesis process of blueberry, the transcriptome analysis was conducted to explore the key genes in blueberry anthocyanin synthesis process.Results: The results showed that ABA could increase the anthocyanin content of blueberry fruits during the veraison. The effect of ABA on blueberry fruit development was systematically analyzed by KEGG and GO. All structural genes and transcription factors (MYB, bHLH and WD40) involved in anthocyanin pathway were identified and their spatiotemporal expression patterns were analyzed. The expression of CHS, CHI, DFR and LDOX / ANS in ABA treated fruits was higher in S5-S6, which was consistent with the change of anthocyanins in fruits.In general, six MYB transcription factors, one bHLH transcription factor and four WD40 transcription factors under treatment were found to have significant changes in transcripts during fruit ripening. Conclusions: Our results suggest that VcMYBA should play a major role in the regulation of anthocyanin synthesis in ABA signaling. This result preliminarily explained the mechanism of ABA increasing anthocyanin content and improves the efficiency of industrial use of blueberry anthocyanins.

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Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Fig (Ficus carica L.)

Frontiers in Plant Science ◽

10.3389/fpls.2021.730692 ◽

2021 ◽

Vol 12 ◽

Author(s):

Miaoyu Song ◽

Haomiao Wang ◽

Zhe Wang ◽

Hantang Huang ◽

Shangwu Chen ◽

...

Keyword(s):

Transcription Factor ◽

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Ficus Carica ◽

Functional Study ◽

Anthocyanin Synthesis ◽

Bhlh Transcription Factor ◽

Transcription Factor Family ◽

Genome Database ◽

Bhlh Genes

The basic helix–loop–helix (bHLH) transcription factor family is the second largest transcription factor family in plants, and participates in various plant growth and development processes. A total of 118 bHLH genes were identified from fig (Ficus carica L.) by whole-genome database search. Phylogenetic analysis with Arabidopsis homologs divided them into 25 subfamilies. Most of the bHLHs in each subfamily shared a similar gene structure and conserved motifs. Seventy-two bHLHs were found expressed at fragments per kilobase per million mapped (FPKM) > 10 in the fig fruit; among them, 15 bHLHs from eight subfamilies had FPKM > 100 in at least one sample. bHLH subfamilies had different expression patterns in the female flower tissue and peel during fig fruit development. Comparing green and purple peel mutants, 13 bHLH genes had a significantly different (≥ 2-fold) expression. Light deprivation resulted in 68 significantly upregulated and 22 downregulated bHLH genes in the peel of the fruit. Sixteen bHLH genes in subfamily III were selected by three sets of transcriptomic data as candidate genes related to anthocyanin synthesis. Interaction network prediction and yeast two-hybrid screening verified the interaction between FcbHLH42 and anthocyanin synthesis-related genes. The transient expression of FcbHLH42 in tobacco led to an apparent anthocyanin accumulation. Our results confirm the first fig bHLH gene involved in fruit color development, laying the foundation for an in-depth functional study on other FcbHLH genes in fig fruit quality formation, and contributing to our understanding of the evolution of bHLH genes in other horticulturally important Ficus species.

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Isolation and Analysis of Anthocyanin Pathway Genes from Ribes Genus Reveals MYB Gene with Potent Anthocyanin-Inducing Capabilities

Plants ◽

10.3390/plants9091078 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1078

Author(s):

Pavel Starkevič ◽

Aušra Ražanskienė ◽

Urtė Starkevič ◽

Vaiva Kazanavičiūtė ◽

Erna Denkovskienė ◽

...

Keyword(s):

Prunus Avium ◽

Anthocyanin Biosynthesis ◽

Regulatory Gene ◽

Anthocyanin Synthesis ◽

Functional Tests ◽

Flavonoid Pathway ◽

Horticultural Crops ◽

Myb Gene ◽

Anthocyanin Pathway ◽

Heterologous System

Horticultural crops of the Ribes genus are valued for their anthocyanin-rich fruits, but until now, there were no data about the genes and regulation of their flavonoid pathway. In this study, the coding sequences of flavonoid pathway enzymes and their putative regulators MYB10, bHLH3 and WD40 were isolated, and their expression analyzed in fruits with varying anthocyanin levels from different cultivars of four species belonging to the Ribes genus. Transcription levels of anthocyanin synthesis enzymes and the regulatory gene RrMYB10 correlated with fruit coloration and anthocyanin quantities of different Ribes cultivars. Regulatory genes were tested for the ability to modulate anthocyanin biosynthesis during transient expression in the leaves of two Nicotiana species and to activate Prunus avium promoters of late anthocyanin biosynthesis genes in N. tabacum. Functional tests showed a strong capability of RrMyb10 to induce anthocyanin synthesis in a heterologous system, even without the concurrent expression of any heterologous bHLH, whereas RrbHLH3 enhanced MYB-induced anthocyanin synthesis. Data obtained in this work facilitate further analysis of the anthocyanin synthesis pathway in key Ribes species, and potent anthocyanin inducer RrMyb10 can be used to manipulate anthocyanin expression in heterologous systems.

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Molecular analysis of anthocyanin-related genes in ornamental cabbage

Genome ◽

10.1139/gen-2017-0098 ◽

2018 ◽

Vol 61 (2) ◽

pp. 111-120 ◽

Cited By ~ 11

Author(s):

Si-Won Jin ◽

Md Abdur Rahim ◽

Hoy-Taek Kim ◽

Jong-In Park ◽

Jong-Goo Kang ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Biosynthetic Genes ◽

Total Anthocyanin ◽

Helix Loop Helix ◽

Anthocyanin Pathway ◽

Total Anthocyanin Content ◽

The Family ◽

Wd40 Repeats

Ornamental cabbage (Brassica oleracea var. acephala) is a winter-grown and important decorative plant of the family Brassicaceae, which displays an exceptional coloration in the central leaves of the rosette. Anthocyanins are the key determinant of the red, purple, and blue colors of vegetative and reproductive parts of many plant species including ornamental cabbage. Total anthocyanin content was measured spectrophotometrically, and the highest anthocyanin content was detected in the red followed by light-red and white ornamental cabbage lines. Anthocyanin biosynthesis is controlled by members of three different transcription factor (TF) families, such as MYB, basic helix-loop-helix (bHLH), and WD40 repeats (WDR), which function as a MBW complex. We identified three MYB, six bHLH, and one WDR TFs that regulate anthocyanin biosynthesis in ornamental cabbage. The expression of the regulatory and biosynthetic genes for anthocyanin synthesis was determined by qPCR. The tested structural genes of the anthocyanin pathway were shown to be up-regulated in the red followed by light-red ornamental cabbage lines; however, the expression levels of the late biosynthetic genes were barely detected in the white ornamental cabbage lines. Among the regulatory genes, BoPAP2 (MYB), BoTT8, BoEGL3.1, and BoMYC1.2 (bHLH), and BoTTG1 (WDR) were identified as candidates for the regulation of anthocyanin biosynthesis. This work could be useful for the breeding of novel colorful ornamental cabbage cultivars.

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FvbHLH9 Functions as a Positive Regulator of Anthocyanin Biosynthesis by Forming a HY5–bHLH9 Transcription Complex in Strawberry Fruits

Plant and Cell Physiology ◽

10.1093/pcp/pcaa010 ◽

2020 ◽

Vol 61 (4) ◽

pp. 826-837 ◽

Cited By ~ 4

Author(s):

Yang Li ◽

Pengbo Xu ◽

Guanqun Chen ◽

Jun Wu ◽

Zhongchi Liu ◽

...

Keyword(s):

Transcription Factor ◽

Promoter Region ◽

Anthocyanin Biosynthesis ◽

Anthocyanin Synthesis ◽

Bhlh Transcription Factor ◽

Anthocyanin Accumulation ◽

Positive Regulator ◽

Key Enzyme ◽

Synergistic Regulation ◽

Transient Overexpression

Abstract Anthocyanin accumulation is transcriptionally regulated by the MYB–bHLH–WD40 complex. Light is indispensable for anthocyanin accumulation, and light-inducible MYB and HY5 were considered to promote anthocyanin accumulation in many fruits. Whether and how light-inducible bHLH transcription factor and HY5 regulate anthocyanin synthesis in strawberry is unknown. In this study, we identified a bHLH transcription factor, FvbHLH9, which was induced by light as well as FvHY5, and found that, similar to FvHY5, the transient overexpression and interference FvbHLH9 in strawberry fruits can promote and decrease anthocyanin accumulation, respectively, indicating FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both FvHY5 and FvbHLH9 specifically bind to the promoter region of some key enzyme genes, including FvDFR, and the expression of FvDFR was activated through the heterodimer formation between FvHY5 and FvbHLH9. Finally, we confirmed that FvbHLH9-promoted anthocyanin accumulation is dependent on HY5–bHLH heterodimerisation in Arabidopsis. Our findings provide insights into a mechanism involving the synergistic regulation of light-dependent coloration and anthocyanin biosynthesis via a HY5–bHLH heterodimer formed by the interaction of FvHY5 and FvbHLH9 in strawberry fruits.

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Transcriptome Co-Expression Network Analysis Identifies Key Genes and Regulators of Sweet Cherry Anthocyanin Biosynthesis

Horticulturae ◽

10.3390/horticulturae7060123 ◽

2021 ◽

Vol 7 (6) ◽

pp. 123

Author(s):

Haiying Yang ◽

Changping Tian ◽

Xiwen Li ◽

Hansheng Gong ◽

Aidi Zhang

Keyword(s):

Transcription Factors ◽

Network Analysis ◽

Sweet Cherry ◽

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Luciferase Assay ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Sweet Cherries ◽

Key Factor

Anthocyanin is the key factor that results in the attractive color of sweet cherry fruits. However, information regarding sweet cherry coloration and the potential mechanisms underlying anthocyanin biosynthesis is limited. In this study, we found that the anthocyanin accumulation varied in sweet cherry flesh and peel, while the anthocyanin content increased sharply in the dark red (DR) stage. Correlations between anthocyanin concentrations and RNA sequencing (RNA-seq), constructed with Weighted Gene Co-Expression Network Analysis (WGCNA), indicated that two structural genes (Pac4CL2, PacANS) and 11 transcription factors (PacbHLH13/74, PacDIV, PacERF109/115, PacGATA8, PacGT2, PacGTE10, PacMYB308, PacPosF21, and PacWRKY7) had similar expression patterns with the changes in anthocyanin content. Additionally, real-time PCR verified all of these gene expression patterns and revealed that PacANS exhibited the highest transcription level. In order to search for potential regulators for anthocyanin biosynthesis, a dual-luciferase assay was performed to investigate the regulatory activities of 11 transcription factors on the PacANS promoter. The results revealed that two novelty bHLHs, PacbHLH13 and PacbHLH74, can trans-activate the PacANS promoter and they might be the candidate genes for regulating anthocyanin synthesis in sweet cherry fruits. The present findings provide a novel viewpoint with regard to anthocyanin biosynthesis mechanisms and the regulatory transcriptional network of fruit coloration in sweet cherries.

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Study on cyanidin metabolism in petals of pink-flowered strawberry based on transcriptome sequencing and metabolite analysis

BMC Plant Biology ◽

10.1186/s12870-019-2048-8 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Li Xue ◽

Jian Wang ◽

Jun Zhao ◽

Yang Zheng ◽

Hai-Feng Wang ◽

...

Keyword(s):

Regulatory Networks ◽

Molecular Mechanisms ◽

Target Genes ◽

Anthocyanin Biosynthesis ◽

Expression Profiles ◽

Expression Patterns ◽

Anthocyanin Synthesis ◽

Integrated Analysis ◽

Intergeneric Hybridization ◽

Significant Differential Expression

Abstract Background Pink-flowered strawberry is a promising new ornamental flower derived from intergeneric hybridization (Fragaria × Potentilla) with bright color, a prolonged flowering period and edible fruits. Its flower color ranges from light pink to red. Pigment compounds accumulated in its fruits were the same as in cultivated strawberry fruits, but different from that in its flowers. However, the transcriptional events underlying the anthocyanin biosynthetic pathway have not been fully characterized in petal coloration. To gain insights into the regulatory networks related to anthocyanin biosynthesis and identify the key genes, we performed an integrated analysis of the transcriptome and metabolome in petals of pink-flowered strawberry. Results The main pigments of red and dark pink petals were anthocyanins, among which cyanidins were the main compound. There were no anthocyanins detected in the white-flowered hybrids. A total of 50,285 non-redundant unigenes were obtained from the transcriptome databases involved in red petals of pink-flowered strawberry cultivar Sijihong at three development stages. Amongst the unigenes found to show significant differential expression, 57 were associated with anthocyanin or other flavonoid biosynthesis, in which they were regulated by 241 differentially expressed members of transcription factor families, such as 40 MYBs, 47 bHLHs, and 41 NACs. Based on a comprehensive analysis relating pigment compounds to gene expression profiles, the mechanism of flower coloration was examined in pink-flowered strawberry. A new hypothesis was proposed to explain the lack of color phenotype of the white-flowered strawberry hybrids based on the transcriptome analysis. The expression patterns of FpDFR and FpANS genes corresponded to the accumulation patterns of cyanidin contents in pink-flowered strawberry hybrids with different shades of pink. Moreover, FpANS, FpBZ1 and FpUGT75C1 genes were the major factors that led to the absence of anthocyanins in the white petals of pink-flowered strawberry hybrids. Meanwhile, the competitive effect of FpFLS and FpDFR genes might further inhibit anthocyanin synthesis. Conclusions The data presented herein are important for understanding the molecular mechanisms underlying the petal pigmentation and will be powerful for integrating novel potential target genes to breed valuable pink-flowered strawberry cultivars.

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Metabolome and Transcriptome Sequencing Analysis Reveals Anthocyanin Metabolism in Pink Flowers of Anthocyanin-Rich Tea (Camellia sinensis)

Molecules ◽

10.3390/molecules24061064 ◽

2019 ◽

Vol 24 (6) ◽

pp. 1064 ◽

Cited By ~ 15

Author(s):

Dylan Rothenberg ◽

Haijun Yang ◽

Meiban Chen ◽

Wenting Zhang ◽

Lingyun Zhang

Keyword(s):

Camellia Sinensis ◽

Flower Development ◽

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Functional Enrichment ◽

Anthocyanin Synthesis ◽

Sequencing Analysis ◽

Anthocyanin Accumulation ◽

Structural Genes ◽

Anthocyanin Pathway

Almost all flowers of the tea plant (Camellia sinensis) are white, which has caused few researchers to pay attention to anthocyanin accumulation and color changing in tea flowers. A new purple-leaf cultivar, Baitang purple tea (BTP) was discovered in the Baitang Mountains of Guangdong, whose flowers are naturally pink, and can provide an opportunity to understand anthocyanin metabolic networks and flower color development in tea flowers. In the present study, twelve anthocyanin components were identified in the pink tea flowers, namely cyanidin O-syringic acid, petunidin 3-O-glucoside, pelargonidin 3-O-beta-d-glucoside, which marks the first time these compounds have been found in the tea flowers. The presence of these anthocyanins seem most likely to be the reason for the pink coloration of the flowers. Twenty-one differentially expressed genes (DEGs) involved in anthocyanin pathway were identified using KEGG pathway functional enrichment, and ten of these DEG’s screened using venn and KEGG functional enrichment analysis during five subsequent stages of flower development. By comparing DEGs and their expression levels across multiple flower development stages, we found that anthocyanin biosynthesis and accumulation in BTP flowers mainly occurred between the third and fourth stages (BTP3 to BTP4). Particularly, during the period of peak anthocyanin synthesis 17 structural genes were upregulated, and four structural genes were downregulated only. Ultimately, eight critical genes were identified using weighted gene co-expression network analysis (WGCNA), which were found to have direct impact on biosynthesis and accumulation of three flavonoid compounds, namely cyanidin 3-O-glucoside, petunidin 3-O-glucoside and epicatechin gallate. These results provide useful information about the molecular mechanisms of coloration in rare pink tea flower of anthocyanin-rich tea, enriching the gene resource and guiding further research on anthocyanin accumulation in purple tea.

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Residues in the WD repeats of Tup1 required for interaction with alpha2.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.6023 ◽

1997 ◽

Vol 17 (10) ◽

pp. 6023-6028 ◽

Cited By ~ 66

Author(s):

K Komachi ◽

A D Johnson

Keyword(s):

Signal Transduction ◽

Amino Acids ◽

Transcriptional Regulation ◽

G Protein ◽

Dna Binding Protein ◽

Beta Subunit ◽

Repeat Proteins ◽

Structural Interface ◽

Wd Repeats ◽

Wd Repeat

The yeast transcriptional repressor Tup1 contains seven WD repeats which interact with the DNA-binding protein alpha2. We have identified mutations in Tup1 that disrupt this interaction. The positions of the amino acids changed by these mutations are consistent with Tup1 being folded into a seven-bladed propeller like that formed by another WD repeat-containing protein, the beta subunit of the heterotrimeric G protein used in signal transduction. Our results also indicate that the interaction between Tup1 and alpha2 resembles the interaction between Gbeta and G alpha, suggesting that a similar structural interface is formed by WD repeat proteins that are used in both transcriptional regulation and signal transduction.

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