Transcriptomic and metabolomic analyses reveal the mechanism of anthocyanin metabolism in H18 pepper leaves and the function of CaDFR1

doi:10.1016/j.jia.2025.10.011

Journal of Integrative Agriculture

2026, Vol. 25

Issue (5): 1949-1960 DOI: 10.1016/j.jia.2025.10.011

Horticulture

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Transcriptomic and metabolomic analyses reveal the mechanism of anthocyanin metabolism in H18 pepper leaves and the function of CaDFR1

Han Wang^{1, 2, 3*}, Dongchen Li^4*, Congsheng Yan^{1, 2, 3}, Muhammad Aamir Manzoor⁵, Qiangqiang Ding^{1, 2, 3}, Yan Wang^{1, 2, 3}, Xiujing Hong^{1, 2, 3}, Tingting Song^{1, 2, 3}, Li Jia^{1, 2, 3#}, Haikun Jiang^{1, 2, 3#}

¹Institute of Vegetables, Anhui Academy of Agricultural Sciences, Hefei 230001, China

² Key Laboratory of Horticultural Crop Germplasm Innovation and Utilization (Co-construction by Ministry and Province), Hefei 230001, China

³ Anhui Provincial Key Laboratory for Germplasm Resources Creation and High-Efficiency Cultivation of Horticultural Crops, Hefei 230001, China

⁴ College of Life Sciences, Anhui Agricultural University, Hefei 230036, China

⁵ School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200030, China

Highlights

● The accumulation of anthocyanins in H18 pepper plants leads to purple leaves, and the anthocyanin content in leaves decreases with plant growth and development.

● Anthocyanins produced with dihydromyricetin (DHM) as substrate are the main component of anthocyanins in H18 pepper leaves.
● CaDFR1 plays an important role in the anthocyanin synthesis pathway of pepper leaves.

Abstract
References

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摘要

花青苷在植物的生长、发育、繁殖和响应胁迫中发挥着至关重要的作用。此外，花青苷的抗氧化特性还能提高水果和蔬菜的品质。虽然前人对花青苷进行了较多的研究，但有关其成分以及花青苷代谢途径基因 DFR（二氢黄酮醇-4-还原酶）在辣椒叶片中的作用的信息还很有限。在这项研究中，我们使用了一种紫色叶片辣椒栽培品种 H18，其叶片上的花青苷随着生长发育而减少。靶向花青素代谢组学检测显示，飞燕草色素、锦葵色素和牵牛花色素的衍生物的含量与花青苷含量呈相同趋势，其中飞燕草色素衍生物是 H18 辣椒叶片的主要成分。对四个不同发育阶段的 H18 叶片进行了转录组测序。结果表明，不同阶段的差异基因主要与生物过程和类黄酮代谢途径有关。通过进化树和转录组表达量分析，确定了3个DFR候选功能基因。底物催化活性分析结果显示，仅CaDFR1表现出对二氢槲皮素(DHQ)、二氢杨梅素(DHM)和二氢山奈酚(DHK)三种底物的催化能力。VIGS 介导的 CaDFR1 沉默导致 H18 辣椒叶片和茎中花青苷含量显著降低，荧光定量显示花青苷代谢途径中其他候选功能基因的表达水平也有所下降。这项研究确定了 H18 辣椒叶片中的关键花青苷成分，并验证了 CaDFR1 的功能，为通过分子育种改变辣椒植株花青素含量提供了理论基础。

Abstract

Anthocyanins play a crucial role in plant growth, development, reproduction, and stress response. Additionally, anthocyanins enhance the quality of fruits and vegetables due to their antioxidant properties. While numerous previous studies have been conducted on anthocyanins, limited information exists regarding their composition and the role of the anthocyanin pathway gene dihydroflavonol 4-reductase (DFR) in chili pepper leaves. In this study, we used a purple leaf pepper cultivar H18 in which the anthocyanin content in leaves decreases with plant growth and development. Targeted anthocyanin metabolite assays revealed that the contents of delphinidin, malvidin, and petunidin derivatives followed the same trend as the overall anthocyanin content, with delphinidin derivatives being the predominant component of H18 pepper leaves. Transcriptome sequencing was performed on H18 leaves at four different stages. The results showed that differentially expressed genes (DEGs) at various stages were primarily associated with biological processes and flavonoid metabolic pathways. Through phylogenetic tree and expression analysis, we identified three candidate genes involved in DFR function. Substrate catalysis assays of CaDFRs demonstrated that only CaDFR1 was active, catalyzing dihydroquercetin (DHQ), dihydromyricetin (DHM), and dihydrokaempferol (DHK). VIGS-mediated silencing of CaDFR1 resulted in a significant decrease in anthocyanin levels in H18 pepper leaves and stems, along with a reduction in the expression levels of other candidate functional genes in the anthocyanin metabolic pathway. This study identifies the key anthocyanin components in the leaves of H18 peppers and validates the function of CaDFR1, providing a theoretical foundation for modifying anthocyanin content in pepper plants through molecular breeding.

Keywords: anthocyanins transcriptomic and metabolomic dihydroflavonol 4-reductase functional validation

Received: 13 February 2025 Accepted: 25 September 2025 Online: 22 October 2025

Fund:

This work was supported by the Postdoctoral Research Program Support of Anhui Province, China (2024C863), the China Agriculture Research System of MOF and MARA (CARS-23-G40, CARS-23-G49), and the Youth Development Fund from Anhui Academy of Agricultural Science, China (QNYC-202121).

About author: #Correspondence Haikun Jiang, Tel/Fax: +86-551-65160817, E-mail: Jhk211@163.com; Li Jia, Tel/Fax: +86-551-65160817, E-mail: jiali820@aaas.org.cn * These authors contributed equally to this study.

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Han Wang, Dongchen Li, Congsheng Yan, Muhammad Aamir Manzoor, Qiangqiang Ding, Yan Wang, Xiujing Hong, Tingting Song, Li Jia, Haikun Jiang. 2026. Transcriptomic and metabolomic analyses reveal the mechanism of anthocyanin metabolism in H18 pepper leaves and the function of CaDFR1. Journal of Integrative Agriculture, 25(5): 1949-1960.

Aiguo Z, Ruiwen D, Cheng W, Cheng C, Dongmei W. 2022. Insights into the catalytic and regulatory mechanisms of dihydroflavonol 4-reductase, a key enzyme of anthocyanin synthesis in Zanthoxylum bungeanum. Tree Physiology, 43, 169–184.

Araguirang G E, Richter A S. 2022. Activation of anthocyanin biosynthesis in high light - what is the initial signal? New Phytologist, 236, 2037–2043.

Baenas N, Belović M, Ilic N, Moreno D A, García-Viguera C. 2019. Industrial use of pepper (Capsicum annum L.) derived products: Technological benefits and biological advantages. Food Chemistry, 274, 872–885.

Bashandy H, Pietiäinen M, Carvalho E, Lim K J, Elomaa P, Martens S, Teeri T H. 2015. Anthocyanin biosynthesis in gerbera cultivar ‘Estelle’ and its acyanic sport ‘Ivory’. Planta, 242, 601–611.

Bate-Smith E C. 1973. Haemanalysis of tannins: The concept of relative astringency. Phytochemistry, 12, 907–912.

Bharti A K, Khurana J P. 2003. Molecular characterization of transparent testa (tt) mutants of Arabidopsis thaliana (ecotype Estland) impaired in flavonoid biosynthetic pathway. Plant Science, 165, 1321–1332.

Burge S, Kelly E, Lonsdale D, Mutowo-Muellenet P, McAnulla C, Mitchell A, Sangrador-Vegas A, Yong S Y, Mulder N, Hunter S. 2012. Manual GO annotation of predictive protein signatures: the InterPro approach to GO curation. Database (Oxford), 2012, bar068.

Butelli E, Titta L, Giorgio M, Mock H P, Matros A, Peterek S, Schijlen E G, Hall R D, Bovy A G, Luo J, Martin C. 2008. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology, 26, 1301–1308.

Cackett L, Luginbuehl L H, Schreier T B, Lopez-Juez E, Hibberd J M. 2022. Chloroplast development in green plant tissues: the interplay between light, hormone, and transcriptional regulation. New Phytologist, 233, 2000–2016.

Cerqueira J V A, de Andrade M T, Rafael D D, Zhu F, Martins S V C, Nunes-Nesi A, Benedito V, Fernie A R, Zsögön A. 2023. Anthocyanins and reactive oxygen species: a team of rivals regulating plant development? Plant Molecular Biology, 112, 213–223.

Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y, Xia R. 2023. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Molecular Plant, 16, 1733–1742.

Chen X, Liu W, Huang X, Fu H, Wang Q, Wang Y, Cao J. 2020. Arg-type dihydroflavonol 4-reductase genes from the fern Dryopteris erythrosora play important roles in the biosynthesis of anthocyanins. PLoS ONE, 15, e0232090.

Davies K M, Landi M, van Klink J W, Schwinn K E, Brummell D A, Albert N W, Chagné D, Jibran R, Kulshrestha S, Zhou Y, Bowman J L. 2022. Evolution and function of red pigmentation in land plants. Annals of Botany, 130, 613–636.

Du M, Zhou K, Liu Y, Deng L, Zhang X, Lin L, Zhou M, Zhao W, Wen C, Xing J, Li C B, Li C. 2020. A biotechnology-based male-sterility system for hybrid seed production in tomato. Plant Journal, 102, 1090–1100.

Espley R V, Jaakola L. 2023. The role of environmental stress in fruit pigmentation. Plant, Cell & Environment, 46, 3663–3679.

Fang K, Liu Y, Wang Z, Zhang X, Zou X, Liu F, Wang Z. 2025. Genome-wide analysis of the CaYABBY family in pepper and functional identification of CaYABBY5 in the regulation of floral determinacy and fruit morphogenesis. Journal of Integrative Agriculture, 24, 3024–3039.

Forkmann G, Ruhnau B J Z f N C. 1987. Distinct substrate specificity of dihydroflavonol 4-reductase from flowers of Petunia hybrida. Zeitschrift fur Naturforschung (C), 42, 1146–1148.

He S, Ye Y, Yuan Y, Lv M, Wang M, Xu Q, Xu X, Chen X. 2023. Insights into flavonoid biosynthesis during cucumber fruit peel coloration based on metabolite profiling and transcriptome analyses. Horticultural Plant Journal, 9, 763–776.

Helariutta Y, Elomaa P, Kotilainen M, Seppänen P, Teeri T H. 1993. Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae). Plant Molecular Biology, 22, 183–193.

Huang Y, Zhou S, Zhao G, Ye F. 2021. Destabilisation and stabilisation of anthocyanins in purple-fleshed sweet potatoes: A review. Trends in Food Science & Technology, 116, 1141–1154.

Jaakola L. 2013. New insights into the regulation of anthocyanin biosynthesis in fruits. Trends Plant Science, 18, 477–483.

Jaakola L, Määttä K, Pirttilä A M, Törrönen R, Kärenlampi S, Hohtola A. 2002. Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiology, 130, 729–739.

Jiang J, Huang H, Gao Q, Li Y, Xiang H, Zeng W, Xu L, Liu X, Li J, Mi Q, Deng L, Yang W, Zhang J, Yang G, Li X. 2023. Effects of editing DFR genes on flowers, leaves, and roots of tobacco. BMC Plant Biology, 23, 349.

Johnson E T, Ryu S, Yi H, Shin B, Cheong H, Choi G. 2001. Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase. Plant Journal, 25, 325–333.

Johnson E T, Yi H, Shin B, Oh B J, Cheong H, Choi G. 1999. Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins. Plant Journal, 19, 81–85.

Kanehisa M, Goto S. 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Research, 28, 27–30.

Kapoor L, Simkin A J, Doss C G P, Siva R. 2022. Fruit ripening: dynamics and integrated analysis of carotenoids and anthocyanins. BMC Plant Biology, 22,

Katsu K, Suzuki R, Tsuchiya W, Inagaki N, Yamazaki T, Hisano T, Yasui Y, Komori T, Koshio M, Kubota S, Walker A R, Furukawa K, Matsui K. 2017. A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity. BMC Plant Biology, 17, 239.

Katsumoto Y, Fukuchi-Mizutani M, Fukui Y, Brugliera F, Holton T A, Karan M, Nakamura N, Yonekura-Sakakibara K, Togami J, Pigeaire A, Tao G Q, Nehra N S, Lu C Y, Dyson B K, Tsuda S, Ashikari T, Kusumi T, Mason J G, Tanaka Y. 2007. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant and Cell Physiology, 48, 1589–1600.

Koes R, Verweij W, Quattrocchio F. 2005. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Science, 10, 236–242.

Kubasek W L, Shirley B W, McKillop A, Goodman H M, Briggs W, Ausubel F M. 1992. Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedlings. The Plant Cell, 4, 1229–1236.

Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874.

Li C, Shi L, Li X, Wang Y, Bi Y, Li W, Ma H, Chen B, Zhu L, Fu Y. 2022. ECAP is a key negative regulator mediating different pathways to modulate salt stress-induced anthocyanin biosynthesis in Arabidopsis. New Phytologist, 233, 2216–2231.

Liu H, Lou Q, Ma J, Su B, Gao Z, Liu Y. 2019. Cloning and functional characterization of dihydroflavonol 4-reductase gene involved in anthocyanidin biosynthesis of grape hyacinth. International Journal of Molecular Sciences, 20,

Liu J, Ai X, Wang Y, Lu Q, Li T, Wu L, Sun L, Shen H. 2020. Fine mapping of the Ca3GT gene controlling anthocyanin biosynthesis in mature unripe fruit of Capsicum annuum L. Theoretical and Applied Genetics, 133, 2729–2742.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method. Methods, 25, 402–408.

Mattioli R, Francioso A, Mosca L, Silva P. 2020. Anthocyanins: A comprehensive review of their chemical properties and health effects on cardiovascular and neurodegenerative diseases. Molecules, 25, E3809.

Mujanović I, Balijagić J, Bajagić M, Poštić D, Đurović S. 2024. Variations in polyphenol content and anthocyanin composition in bilberry populations (Vaccinium myrtillus L.) due to environmental factors. Journal of Food Composition and Analysis, 136, 106732.

Nakatsuka T, Suzuki T, Harada K, Kobayashi Y, Dohra H, Ohno H. 2019. Floral organ- and temperature-dependent regulation of anthocyanin biosynthesis in Cymbidium hybrid flowers. Plant Science, 287, 110173.

Petit P, Granier T, d’Estaintot B L, Manigand C, Bathany K, Schmitter J-M, Lauvergeat V, Hamdi S, Gallois B. 2007. Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis. Journal of Molecular Biology, 368, 1345–1357.

Petroni K, Pilu R, Tonelli C. 2014. Anthocyanins in corn: A wealth of genes for human health. Planta, 240, 901–911.

Polashock J J, Griesbach R J, Sullivan R F, Vorsa N. 2002. Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco. Plant Science, 163, 241–251.

Rodríguez-Lorenzo M, Mauri N, Royo C, Rambla J L, Diretto G, Demurtas O, Hilbert G, Renaud C, Tobar V, Huete J, Delrot S, Granell A, Martínez-Zapater J M, Carbonell-Bejerano P. 2023. The flavour of grape colour: anthocyanin content tunes aroma precursor composition by altering the berry microenvironment. Journal of Experimental Botany, 74, 6369–6390.

Rodríguez-Mena A, Ochoa-Martínez L A, González-Herrera S M, Rutiaga-Quiñones O M, González-Laredo R F, Olmedilla-Alonso B. 2023. Natural pigments of plant origin: Classification, extraction and application in foods. Food Chemistry, 398,

Ruan H, Shi X, Gao L, Rashid A, Li Y, Lei T, Dai X, Xia T, Wang Y. 2022. Functional analysis of the dihydroflavonol 4-reductase family of Camellia sinensis: exploiting key amino acids to reconstruct reduction activity. Horticulture Research, 9, uhac098.

Saigo T, Wang T, Watanabe M, Tohge T. 2020. Diversity of anthocyanin and proanthocyanin biosynthesis in land plants. Current Opinion In Plant Biology, 55, 93–99.

Seitz C, Vitten M, Steinbach P, Hartl S, Hirsche J, Rathje W, Treutter D, Forkmann G. 2007. Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy. Phytochemistry, 68, 824–833.

Shi L, Li X, Fu Y, Li C. 2023. Environmental stimuli and phytohormones in anthocyanin biosynthesis: A comprehensive review. International Journal of Molecular Sciences, 24, 16415.

Shi Q, Du J, Zhu D, Li X, Li X. 2020. Metabolomic and transcriptomic analyses of anthocyanin biosynthesis mechanisms in the color mutant Ziziphus jujuba cv. Tailihong. Journal of Agricultural and Food Chemistry, 68, 15186–15198.

Sun C, Deng L, Du M, Zhao J, Chen Q, Huang T, Jiang H, Li C B, Li C. 2020. A transcriptional network promotes anthocyanin biosynthesis in tomato flesh. Molecular Plant, 13, 42–58.

Sun H L, Wang X Y, Shang Y, Wang X Q, Du G D, LÜ D G. 2021. Preharvest application of melatonin induces anthocyanin accumulation and related gene upregulation in red pear (Pyrus ussuriensis). Journal of Integrative Agriculture, 20, 2126–2137.

Tanaka Y, Brugliera F, Chandler S. 2009. Recent progress of flower colour modification by biotechnology. International Journal of Molecular Sciences, 10, 5350–5369.

Tian J, Chen M C, Zhang J, Li K T, Song T T, Zhang X, Yao Y C. 2017. Characteristics of dihydroflavonol 4-reductase gene promoters from different leaf colored Malus crabapple cultivars. Horticulture Research, 4, 17070.

Wan H, Yuan W, Ruan M, Ye Q, Wang R, Li Z, Zhou G, Yao Z, Zhao J, Liu S, Yang Y. 2011. Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L.). Biochemical and Biophysical Research Communications, 416, 24–30.

Wang G, Chen B, Du H, Zhang F, Zhang H, Wang Y, He H, Geng S, Zhang X. 2018. Genetic mapping of anthocyanin accumulation-related genes in pepper fruits using a combination of SLAF-seq and BSA. PLoS ONE, 13, e0204690.

Wang X, Chen X, Luo S, Ma W, Li N, Zhang W, Tikunov Y, Xuan S, Zhao J, Wang Y, Zheng G, Yu P, Bai Y, Bovy A, Shen S. 2022. Discovery of a DFR gene that controls anthocyanin accumulation in the spiny Solanum group: roles of a natural promoter variant and alternative splicing. Plant Journal, 111, 1096–1109.

Xia D, Zhou H, Wang Y, Li P, Fu P, Wu B, He Y. 2021. How rice organs are colored: The genetic basis of anthocyanin biosynthesis in rice. The Crop Journal, 9, 598–608.

Xie D Y, Jackson L A, Cooper J D, Ferreira D, Paiva N L. 2004. Molecular and biochemical analysis of two cDNA clones encoding dihydroflavonol-4-reductase from Medicago truncatula. Plant Physiology, 134, 979–994.

Xu H, Wang G, Ji X, Xiang K, Wang T, Zhang M, Shen G, Zhang R, Zhang J, Chen X. 2024. JrATHB-12 mediates JrMYB113 and JrMYB27 to control the anthocyanin levels in different types of red walnut. Journal of Integrative Agriculture, 23, 2649–2661.

Ying J, Wen S, Cai Y, Ye Y, Li L, Qian R. 2024. Decoding anthocyanin biosynthesis regulation in Asparagus officinalis peel coloration: Insights from integrated metabolomic and transcriptomic analyses. Plant Physiology and Biochemistry, 215, 108980.

Zhang J, Li S, An H, Zhang X, Zhou B. 2022. Integrated transcriptome and metabolome analysis reveals the anthocyanin biosynthesis mechanisms in blueberry (Vaccinium corymbosum L.) leaves under different light qualities. Frontiers in Plant Science, 13, 1073332.

Zhang S L, Deng P, Xu Y C, Lü S W, Wang J Q. 2016. Quantification and analysis of anthocyanin and flavonoids compositions, and antioxidant activities in onions with three different colors. Journal of Integrative Agriculture, 15, 2175–2181.

Zhang Y, Butelli E, Martin C. 2014. Engineering anthocyanin biosynthesis in plants. Current Opinion in Plant Biology, 19, 81–90.

Zhou Y, Deng Y, Liu D, Wang H, Zhang X, Liu T, Wang J, Li Y, Ou L, Liu F, Zou X, Ouyang B, Li F. 2021. Promoting virus-induced gene silencing of pepper genes by a heterologous viral silencing suppressor. Plant Biotechnology Journal, 19, 2398–2400.

Zhu L, Liao Y, Zhang T, Zeng Z, Wang J, Duan L, Chen X, Lin K, Liang X, Han Z, Huang Y, Wu W, Hu H, Xu Z F, Ni J. 2024. Reactive oxygen species act as the key signaling molecules mediating light-induced anthocyanin biosynthesis in Eucalyptus. Plant Physiology and Biochemistry, 212, 108715.

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