Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in red walnut

Abstract Background Walnut is one of the most important dry fruit crops worldwide, typically green leaves and yellow-brown or gray-yellow seed coats. A specific walnut type, red walnut ‘RW-1’ with red leaves and seed coats was selected as plant material because of higher anthocyanins contents. Anthocyanins are important colorants with strong antioxidant activity, especially, benefic for human health. However, few studies focused on the molecular mechanism of anthocyanin biosynthesis in walnut. Results From the results of Widely Targeted Metabolome and anthocyanidin detection analysis, 395 substances, including 4 procyanidins and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all the anthocyanins in SR were significantly up-accumulated comparing with SG. Also, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified to the primary components of anthocyanidins because of the higher contents. It was noted that 9 anthocyanins including malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside) were detected only in SR walnut. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H and UFGTs), and four MYBs in anthocyanin biosynthetic pathway were significantly higher in SR walnut. Conclusions We identified the color formation of SR leaves is due to the accumulation of anthocyanins. And our results obtained the valuable information on the anthocyanin metabolites and candidate genes in anthocyanin biosynthesis, which provided new insights into the anthocyanin biosynthesis in walnuts.

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Integrated Metabolomic and Transcriptomic Analysis of the Anthocyanin and Proanthocyanin Regulatory Networks in Red Walnut Natural Hybrid Progeny Leaves

10.21203/rs.3.rs-1089011/v1 ◽

2021 ◽

Author(s):

Lei Wang ◽

Lin Li ◽

Wei Zhao ◽

Haijun Meng ◽

Ganggang Zhang ◽

...

Keyword(s):

Regulatory Networks ◽

Anthocyanin Biosynthesis ◽

Natural Hybrid ◽

Structural Genes ◽

Fruit Crops ◽

Biotic And Abiotic Stresses ◽

Yellow Seed ◽

Detection Analysis ◽

Dry Fruit ◽

Seed Coats

Abstract BackgroundWalnuts are one of the most important dry fruit crops worldwide, typically exhibiting green leaves and yellow–brown or gray–yellow seed coats. A specific walnut type, red walnut ‘RW-1’, with red leaves and seed coats was selected as the plant material because of its higher anthocyanin and proanthocyanin (PA) contents. Anthocyanins and PAs coprise important secondary defense methods for plants to respond to biotic and abiotic stresses. However, few studies have focused on the molecular mechanism of anthocyanin biosynthesis in walnuts.ResultsFrom the results of widely targeted metabolome and anthocyanidin detection analysis, 395 substances, including 4 PAs and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all anthocyanin types in SR were significantly upregulated compared with SG. Additionally, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified as the primary components of anthocyanidins because of their higher contents. Nine anthocyanidins, malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside), were detected only in the SR walnuts. For PAs, proanthocyanin C1 was upregulated in SR compared with SG, while proanthocyanin B1 and proanthocyanin B3 were upregulated in SR-1 and SR-3 but downregulated in SR-2 compared with the controls. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H, UFGTs, LAR and ANR), four MYBs and six WD40s in the anthocyanin and PA biosynthetic pathways were significantly higher in the SR walnut.ConclusionsOur results provide valuable information on anthocyanin and PA metabolites and candidate genes in anthocyanin and PA biosynthesis, which provides new insights into anthocyanin and PA biosynthesis in walnuts.

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Comparative transcriptome analysis reveals key genes associated with pigmentation in radish (Raphanus sativus L.) skin and flesh

Scientific Reports ◽

10.1038/s41598-021-90633-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jifang Zhang ◽

Jian Zhao ◽

Qunyun Tan ◽

Xiaojun Qiu ◽

Shiyong Mei

Keyword(s):

Transcription Factors ◽

Raphanus Sativus ◽

Regulatory Networks ◽

Anthocyanin Biosynthesis ◽

Comparative Transcriptome ◽

Structural Genes ◽

Key Genes ◽

Transcript Profiles ◽

Species Specific ◽

Anthocyanin Biosynthetic Genes

AbstractRadish (Raphanus sativus) is an important vegetable worldwide that exhibits different flesh and skin colors. The anthocyanins responsible for the red and purple coloring in radishes possess nutritional value and pharmaceutical potential. To explore the structural and regulatory networks related to anthocyanin biosynthesis and identify key genes, we performed comparative transcriptome analyses of the skin and flesh of six colored radish accessions. The transcript profiles showed that each accession had a species-specific transcript profile. For radish pigmentation accumulation, the expression levels of anthocyanin biosynthetic genes (RsTT4, RsC4H, RsTT7, RsCCOAMT, RsDFR, and RsLDOX) were significantly upregulated in the red- and purple-colored accessions, but were downregulated or absent in the white and black accessions. The correlation test, combined with metabolome (PCC > 0.95), revealed five structural genes (RsTT4, RsDFR, RsCCOAMT, RsF3H, and RsBG8L) and three transcription factors (RsTT8-1, RsTT8-2, and RsPAR1) to be significantly correlated with flavonoids in the skin of the taproot. Four structural genes (RsBG8L, RsDFR, RsCCOAMT, and RsLDOX) and nine transcription factors (RsTT8-1, RsTT8-2, RsMYB24L, RsbHLH57, RsPAR2L, RsbHLH113L, RsOGR3L, RsMYB24, and RsMYB34L) were found to be significantly correlated with metabolites in the flesh of the taproot. This study provides a foundation for future studies on the gene functions and genetic diversity of radish pigmentation and should aid in the cultivation of new valuable radish varieties.

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Co-Expression Network Analyses of Anthocyanin Biosynthesis Genes in Ruellia (Wild Petunias; Acanthaceae)

10.21203/rs.3.rs-58072/v1 ◽

2020 ◽

Author(s):

Erin Tripp ◽

Yongbin Zhuang

Keyword(s):

Regulatory Networks ◽

Anthocyanin Biosynthesis ◽

Purifying Selection ◽

Flower Color ◽

Molecular Architecture ◽

Structural Genes ◽

Sequence Comparisons ◽

Network Analyses ◽

Regulatory Loci ◽

Rates Of Molecular Evolution

Abstract Background. Anthocyanins are major pigments contributing to flower coloration and as such knowledge of molecular architecture underlying the anthocyanin biosynthetic pathway (ABP) is key to understanding flower color diversification. To identify ABP structural genes and associated regulatory networks, we sequenced 16 transcriptomes generated from 10 species of Ruellia and then conducted co-expression analyses among resulting data. Results. Complete coding sequences for 12 candidate structural loci representing eight genes plus nine candidate regulatory loci were assembled. Analysis of non-synonymous/synonymous (dn/ds) mutation rates indicated all identified loci are under purifying selection, suggesting overall selection to prevent the accumulation of deleterious mutations. Additionally, upstream enzymes have lower rates of molecular evolution compared to downstream enzymes. However, site-specific tests of selection yielded evidence for positive selection at several sites, including four in F3'H2 and five in DFR3, and these sites are located in protein binding regions. A species-level phylogenetic tree constructed using a newly implemented hybrid transcriptome–RADseq approach implicates numerous flower color transitions among the 10 species. We found evidence of both regulatory and structural mutations to F3'5'H in helping to explain the evolution of red flowers from purple-flowered ancestors.Conclusions. Sequence comparisons and co-expression analyses of ABP loci revealed that mutations in regulatory loci are likely to play a greater role in flower color transitions in Ruellia compared to mutations in underlying structural genes.

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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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Analysis of structural genes and key transcription factors related to anthocyanin biosynthesis in potato tubers

Scientia Horticulturae ◽

10.1016/j.scienta.2017.07.018 ◽

2017 ◽

Vol 225 ◽

pp. 310-316 ◽

Cited By ~ 10

Author(s):

Huiling Zhang ◽

Bo Yang ◽

Jun Liu ◽

Dalong Guo ◽

Juan Hou ◽

...

Keyword(s):

Transcription Factors ◽

Anthocyanin Biosynthesis ◽

Potato Tubers ◽

Structural Genes

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Combined Transcriptome and Metabolome Integrated Analysis of Acer mandshuricum to Reveal Candidate Genes Involved in Anthocyanin Accumulation

10.21203/rs.3.rs-796758/v1 ◽

2021 ◽

Author(s):

Shikai Zhang ◽

Wang Zhan ◽

Anran Sun ◽

Ying Xie ◽

Zhiming Han ◽

...

Keyword(s):

Regulatory Mechanism ◽

Color Change ◽

Anthocyanin Biosynthesis ◽

Integrated Analysis ◽

Anthocyanin Accumulation ◽

Structural Genes ◽

Leaf Color ◽

Accumulation Pattern ◽

Color Formation ◽

Red Color

Abstract The red color formation of Acer mandshuricum leaves is caused by the accumulation of anthocyanins primarily, but the molecular mechanism researches which underlie anthocyanin biosynthesis in A. mandshuricum were still lacking. Therefore, we combined the transcriptome and metabolome and analyzed the regulatory mechanism and accumulation pattern of anthocyanins in leaf color change periods in three different leaf color states. In our results, 26 anthocyanins were identified. Notably, the metabolite cyanidin 3-O-glucoside was found that significantly correlated with the color formation, was the predominant metabolite in anthocyanin biosynthesis of A. mandshuricum. By the way, two key structural genes ANS (Cluster-20561.86285) and BZ1 (Cluster-20561.99238) in anthocyanidin biosynthesis pathway were significantly up-regulated in RL, suggesting that they might enhance accumulation of cyanidin 3-O-glucoside which is their downstream metabolite, and contributed the red formation of A. mandshuricum leaves. Additionally, most TFs (e.g., MYBs, bZIPs and bHLHs) were detected differentially expressed in three leaf color stages that could participate in anthocyanin accumulation. This study sheds light on the anthocyanin molecular regulation of anthocyanidin bio-synthesis and accumulation underlying the different leaf color change periods in A. mandshuricum, and it could provide basic theory and new insight for the leaf color related genetic improvement of A. mandshuricum.

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The Flavonoid Biosynthesis Network in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms222312824 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12824

Author(s):

Weixin Liu ◽

Yi Feng ◽

Suhang Yu ◽

Zhengqi Fan ◽

Xinlei Li ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Current Knowledge ◽

Anthocyanin Biosynthesis ◽

Basic Structure ◽

Ring Structure ◽

Flavonoid Biosynthesis ◽

Comprehensive Overview ◽

Flavonoid Biosynthetic Pathway ◽

Intermediate Metabolites ◽

Important Intermediate

Flavonoids are an important class of secondary metabolites widely found in plants, contributing to plant growth and development and having prominent applications in food and medicine. The biosynthesis of flavonoids has long been the focus of intense research in plant biology. Flavonoids are derived from the phenylpropanoid metabolic pathway, and have a basic structure that comprises a C15 benzene ring structure of C6-C3-C6. Over recent decades, a considerable number of studies have been directed at elucidating the mechanisms involved in flavonoid biosynthesis in plants. In this review, we systematically summarize the flavonoid biosynthetic pathway. We further assemble an exhaustive map of flavonoid biosynthesis in plants comprising eight branches (stilbene, aurone, flavone, isoflavone, flavonol, phlobaphene, proanthocyanidin, and anthocyanin biosynthesis) and four important intermediate metabolites (chalcone, flavanone, dihydroflavonol, and leucoanthocyanidin). This review affords a comprehensive overview of the current knowledge regarding flavonoid biosynthesis, and provides the theoretical basis for further elucidating the pathways involved in the biosynthesis of flavonoids, which will aid in better understanding their functions and potential uses.

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CBFs Function in Anthocyanin Biosynthesis by Interacting with MYB113 in Eggplant (Solanum melongena L.)

Plant and Cell Physiology ◽

10.1093/pcp/pcz209 ◽

2019 ◽

Vol 61 (2) ◽

pp. 416-426 ◽

Cited By ~ 1

Author(s):

Lu Zhou ◽

Yongjun He ◽

Jing Li ◽

Yang Liu ◽

Huoying Chen

Keyword(s):

Low Temperature ◽

Anthocyanin Biosynthesis ◽

Solanum Melongena ◽

Structural Genes ◽

Cold Response ◽

Anthocyanin Pathway ◽

R2r3 Myb ◽

Dependent Pathway ◽

Shed Light ◽

R2r3 Myb Transcription Factors

Abstract Eggplant is rich in anthocyanins. R2R3-MYB transcription factors play a key role in the anthocyanin pathway. Low temperature is vital abiotic stress that affects the anthocyanin biosynthesis in plants. CBFs (C-repeat binding factors) act as central regulators in cold response. In this study, we found that SmCBF1, SmCBF2 and SmCBF3, via their C-terminal, physically interacted with SmMYB113, a key regulator of anthocyanin biosynthesis in eggplant. SmCBF2 and SmCBF3 upregulated the expression of SmCHS and SmDFR via a SmMYB113-dependent pathway. In addition, the transient expression assays demonstrated that co-infiltrating SmCBFs and SmMYB113 significantly improved the contents of anthocyanin and the expression levels of anthocyanin structural genes in tobacco. When SmTT8, a bHLH partner of SmMYB113, coexpressed with SmCBFs and SmMYB113, the anthocyanin contents were significantly enhanced compared with SmCBFs and SmMYB113. Furthermore, overexpression of SmCBF2 and SmCBF3 could facilitate the anthocyanin accumulation under cold conditions in Arabidopsis. Taken together, these results shed light on the functions of SmCBFs and potential mechanisms of low-temperature-induced anthocyanin biosynthesis in eggplant.

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iTRAQ-Based Protein Profiling Provides Insights into the Mechanism of Light-Induced Anthocyanin Biosynthesis in Chrysanthemum (Chrysanthemum × morifolium)

Genes ◽

10.3390/genes10121024 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1024

Author(s):

Yan Hong ◽

Mengling Li ◽

Silan Dai

Keyword(s):

Biosynthetic Pathway ◽

Developmental Stages ◽

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Flower Color ◽

Chrysanthemum Morifolium ◽

Light Signal ◽

Protein Profiling ◽

Differentially Expressed ◽

Anthocyanin Biosynthetic Pathway

The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis, but the deep molecular mechanism remains elusive. In our previous study, a series of light-induced structural and regulatory genes involved in the anthocyanin biosynthetic pathway in the chrysanthemum were identified using RNA sequencing. In the present study, differentially expressed proteins that are in response to light with the capitulum development of the chrysanthemum ‘Purple Reagan’ were further identified using isobaric tags for relative and absolute quantification (iTRAQ) technique, and correlation between the proteomic and the transcriptomic libraries was analyzed. In general, 5106 raw proteins were assembled based on six proteomic libraries (three capitulum developmental stages × two light treatments). As many as 160 proteins were differentially expressed between the light and the dark libraries with 45 upregulated and 115 downregulated proteins in response to shading. Comparative analysis between the pathway enrichment and the gene expression patterns indicated that most of the proteins involved in the anthocyanin biosynthetic pathway were downregulated after shading, which was consistent with the expression patterns of corresponding encoding genes; while five light-harvesting chlorophyll a/b-binding proteins were initially downregulated after shading, and their expressions were enhanced with the capitulum development thereafter. As revealed by correlation analysis between the proteomic and the transcriptomic libraries, GDSL esterase APG might also play an important role in light signal transduction. Finally, a putative mechanism of light-induced anthocyanin biosynthesis in the chrysanthemum was proposed. This study will help us to clearly identify light-induced proteins associated with flower color in the chrysanthemum and to enrich the complex mechanism of anthocyanin biosynthesis for use in cultivar breeding.

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Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color

International Journal of Molecular Sciences ◽

10.3390/ijms21082901 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2901 ◽

Cited By ~ 1

Author(s):

Jana Jeevan Rameneni ◽

Su Ryun Choi ◽

Sushil Satish Chhapekar ◽

Man-Sun Kim ◽

Sonam Singh ◽

...

Keyword(s):

Chinese Cabbage ◽

Anthocyanin Biosynthesis ◽

Gene Promoters ◽

Biosynthesis Pathway ◽

Transcriptome Data ◽

Biosynthetic Gene ◽

Structural Genes ◽

Regulatory Motifs ◽

Myb Genes ◽

Anthocyanin Biosynthetic Genes

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC.

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