Functional Study of RcMYB4 in Regulating Anthocyanin Biosynthesis in Rehmannia chingii

doi:10.16420/j.issn.0513-353x.2025-0309

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (3): 735-748.doi: 10.16420/j.issn.0513-353x.2025-0309

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Functional Study of RcMYB4 in Regulating Anthocyanin Biosynthesis in Rehmannia chingii

YANG Yahe¹, DING Ning¹, HAO Qiuwen¹, GAO Junge¹, ZHANG Pengyu¹, HU Jingyi¹, ZHANG Zhongyi², WANG Fengqing¹^,^**()

¹ College of Agronomy，Henan Agricultural University，Zhengzhou 450046，China
² College of Agriculture，Fujian Agriculture and Forestry University，Fuzhou 350002，China

Received:2025-05-25 Revised:2025-10-23 Online:2026-03-25 Published:2026-03-20
Contact: WANG Fengqing

Abstract

Abstract:

Using Rehmannia chingii as the experimental material，a novel R2R3-MYB transcription factor gene was cloned from its corolla，with the resulting gene being systematically named RcMYB4. The open reading frame（ORF）of the gene spans 777 bp，encoding 258 amino acids residues. Quantitative real-time PCR（qRT-PCR）analysis demonstrated predominant expression of RcMYB4 in the corolla tube. Multiple sequence alignment of protein homologs revealed that RcMYB4 contain conserved domains of the S6 subfamily. Phylogenetic tree construction using the neighbor-joining method revealed that RcMYB4 groups with RcMYB1 from R. chingii and RgMYB42 from R. glutinosa，exhibiting the closest phylogenetic relationship. Transient expression assays in Nicotiana benthamiana indicated that heterologous expression of RcMYB4 activated anthocyanin biosynthesis，with co-expression of RcMYB4 + RcANS significantly enhanced anthocyanin accumulation compared to single transformation of RcMYB4. Agrobacterium tumefaciens-mediated genetic transformation in R. glutinosa revealed that transgenic lines overexpressing RcMYB4 exhibited significantly increased anthocyanin accumulation in both callus tissues and leaves of tissue-cultured seedlings compared to wild-type controls. In potted nutrient soil-grown transgenic R. glutinosa，both leaves and petioles exhibited significantly increased anthocyanin accumulation compared to wild-type controls. Notably，only the OE7 transgenic line showed enhanced anthocyanin content in tuberous roots，reaching 4.01 times that of the wild-type control. Expression analysis of anthocyanin biosynthetic structural genes in RcMYB4-overexpressing R. glutinosa lines revealed significant upregulation of RgANS，RgCHS，RgF3H，and RgDFR in leaves across three transgenic lines，with OE7 exhibiting the most pronounced increases. In tuberous roots，these four structural genes were exclusively upregulated in OE7. These results demonstrate that RcMYB4 is a positive regulatory transcription factor that activates anthocyanin biosynthesis in Rehmannia chingii.

Key words: Rehmannia chingii, RcMYB4, Rehmannia glutinosa, anthocyanin, heterologous expression

YANG Yahe, DING Ning, HAO Qiuwen, GAO Junge, ZHANG Pengyu, HU Jingyi, ZHANG Zhongyi, WANG Fengqing. Functional Study of RcMYB4 in Regulating Anthocyanin Biosynthesis in Rehmannia chingii[J]. Acta Horticulturae Sinica, 2026, 53(3): 735-748.

Figures/Tables 8

Fig. 1 PCR amplification of RcMYB4（A）and its expression in different tissues of Rehmannia chingii（B）

Fig. 2 Multiplesequence alignmentand domains of RcMYB4 withits homologous proteins Rh：Rehmannia henryi；Rg：Rehmannia glutinosa；Rc：Rehmannia chingii；Rp：Rehmannia piasezkii；At：Arabidopsis thaliana

Fig. 3 Phylogenetic tree of RcMYB4 and its homologous proteins

Fig. 4 Phenotypic analysis of transient overexpression of RcMYB4 in Nicotiana benthamiana A：Transient expression of RcMYB4 and RcMYB4 + RcANS in Nicotiana benthamiana leaves phenotype；B：Detection of total anthocyanin content in Nicotiana benthamiana leaves. ** P < 0.01. The same below

Fig. 5 Overexpression of RcMYB4 in callus induction and morphology（A）and anthocyanin content analysis（B）of Rehmannia glutinosa WT：Wild type；OE：Overexpression lines

Fig. 6 Phenotype（A）and anthocyanin content analysis（B）of RcMYB4-overexpressing Rehmannia glutinosa tissue-cultured seedlings

Fig. 7 Phenotype and anthocyanin content analysis of RcMYB4-overexpressing Rehmannia glutinosa plants A：The phenotype of Rehmannia glutinosa plants overexpressing RcMYB4；B：Cross-sectional morphology of leaves，mesophyll，and petioles in RcMYB4-overexpressing Rehmannia glutinosa plants；C：The tuberous roots and their cross-sectional morphology of RcMYB4-overexpressing Rehmannia glutinosa；D-F：Anthocyanin content in leaves，petioles，and tuberous roots of different transgenic lines

Fig. 8 Expression levels of structural genes in the anthocyanin biosynthesis pathway in leaves（A）and tuberous roots（B）of Rehmannia glutinosa stably expressing RcMYB4

References 28

[1]	Ahn J Y, Kim J, Yang J Y, Lee H J, Kim S, Cho K S, Lee S H, Kim J H, Lee T H, Hur Y, Shim D. 2022. Comparative transcriptome analysis between two potato cultivars in tuber induction to reveal associated genes with anthocyanin accumulation. International Journal of Molecular Sciences, 23 (7):3681. doi: 10.3390/ijms23073681 URL
[2]	Buhrman K, Aravena-Calvo J, Ross Zaulich C, Hinz K, Laursen T. 2022. Anthocyanic vacuolar inclusions:from biosynthesis to storage and possible applications. Frontiers in Chemistry,10:913324.
[3]	Chen K, Du L, Liu H, Liu Y. 2019. A novel R2R3-MYB from grape hyacinth,MaMybA,which is different from MaAN2,confers intense and magenta anthocyanin pigmentation in tobacco. BMC Plant Biology, 19 (1):390. doi: 10.1186/s12870-019-1999-0
[4]	Chen Y N, Shao G Y, Chen L M, Zhang H Y, Cui Y Y, Luo P. 2025. A feedback loop comprising RhMYB114 and RhMYB16 regulates anthocyanin accumulation and tissue acidification in Rosa hybrida. Horticultural Plant Journal, 11 (6):2194-2210. doi: 10.1016/j.hpj.2024.07.004 URL
[5]	Damar I, Ekşi A. 2012. Antioxidant capacity and anthocyanin profile of sour cherry(Prunus cerasus L.)juice. Food Chemistry, 135 (4):2910-2914. doi: 10.1016/j.foodchem.2012.07.032 pmid: 22980889
[6]	Deng Y R, Yang J W, Wei J J, Liu S Y, Yang L, Wang X F, Zhang N, Si H J. 2025. stu-miR159a negatively regulates anthocyanin-specific MYB transcription factor to mediate drought stress tolerance in potato. Horticultural Plant Journal, 11 (5):1917-1929. doi: 10.1016/j.hpj.2024.03.016 URL
[7]	Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. 2010. MYB transcription factors in Arabidopsis. Trends in Plant Science, 15 (10):573-581. doi: 10.1016/j.tplants.2010.06.005 URL
[8]	Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. The Plant Journal, 53 (5):814-827. doi: 10.1111/j.1365-313X.2007.03373.x pmid: 18036197
[9]	Huang Jiake, Hong Sidan, Xu Haihua, Gu Cuihua. 2025. Progress on the formation mechanism of Lagerstroemia indica flower color. Acta Horticulturae Sinica, 52 (12):3412-3426. (in Chinese)
	黄嘉可, 洪思丹, 徐海华, 顾翠花. 2025. 紫薇花色呈色机制研究进展. 园艺学报, 52 (12):3412-3426. doi: 10.16420/j.issn.0513-353x.2024-1021
[10]	Ji X, Li Z, Zhang M, Lang S, Song X. 2024. ChMYB1-ChbHLH42-ChTTG 1 module regulates abscisic acid-induced anthocyanin biosynthesis in Cerasus humilis. Horticultural Plant Journal, 10 (1):51-65. doi: 10.1016/j.hpj.2023.05.015 URL
[11]	Li P, Li Y J, Zhang F J, Zhang G Z, Jiang X Y, Yu H M, Hou B K. 2017. The Arabidopsis UDP-glycosyltransferases UGT79B2 and UGT79B3,contribute to cold,salt and drought stress tolerance via modulating anthocyanin accumulation. The Plant Journal, 89 (1):85-103. doi: 10.1111/tpj.2017.89.issue-1 URL
[12]	Li Y, Li M, Guo Z, Liu J, Chen P, Lu W, Jiang C, Xiao J, Lei F, Zheng Y. 2025. AoMYB114 transcription factor regulates anthocyanin biosynthesis in the epidermis of tender asparagus stems. Frontiers in Plant Science,16:1531574.
[13]	Liu X, Liu H, Tian B, Shi G, Liu C, Guo J, Cao G, Wei F. 2023. Metabolome and transcriptome analyses of anthocyanin biosynthesis reveal key metabolites and candidate genes in purple wheat(Triticum aestivum L.). Physiologia Plantarum, 175 (3):e13921. doi: 10.1111/ppl.v175.3 URL
[14]	Miao C, Song C, Ding N, Zuo X, Zhang Z, Zhang X, Mu J, Wang F. 2024. De novo transcriptome analysis identifies RpMYB1 as an activator of anthocyanin biosynthesis in Rehmannia piasezkii. Plant Physiology and Biochemistry,215:108964.
[15]	Montefiori M, Brendolise C, Dare A PmLin-Wang K, Davies K M, Hellens R P, Allan A C. 2015. In the Solanaceae,a hierarchy of bHLHs confer distinct target specificity to the anthocyanin regulatory complex. Journal Experimental Botany, 66 (5):1427-1436. doi: 10.1093/jxb/eru494 URL
[16]	Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R. 1999. Molecular analysis of the anthocyanin 2 gene of petunia and its role in the evolution of flower color. The Plant Cell, 11 (8):1433-1444. doi: 10.1105/tpc.11.8.1433 URL
[17]	Tang Q, Chi F M, Liu H D, Zhang H J, Song Y. 2021. Single-molecule real-time and Illumina sequencing to analyze transcriptional regulation of flavonoid synthesis in blueberry. Frontiers in Plant Science,12:754325.
[18]	Wu H, Pan Y, Ni E, Qin D, Fang K, Wang Q, Yang C, Luo M, Liu J. 2024. CsRAB,a R2R3-MYB transcription factor from purple tea(Camellia sinensis),positively regulates anthocyanin biosynthesis. Frontiers in Plant Science,15:1514631.
[19]	Xu Z S, Feng K, Que F, Wang F, Xiong A S. 2017. A MYB transcription factor,DcMYB6,is involved in regulating anthocyanin biosynthesis in purple carrot taproots. Scientific Reports,7:45324.
[20]	Xu Z S, Yang Q Q, Feng K, Yu X, Xiong A S. 2020. DcMYB113,a root-specific R2R3-MYB,conditions anthocyanin biosynthesis and modification in carrot. Plant Biotechnology Journal, 18 (7):1585-1597. doi: 10.1111/pbi.v18.7 URL
[21]	Yan H, Pei X, Zhang H, Li X, Zhang X, Zhao M, Chiang V L, Sederoff R R, Zhao X. 2021. MYB-mediated regulation of anthocyanin biosynthesis. International Journal of Molecular Sciences, 22 (6):3103. doi: 10.3390/ijms22063103 URL
[22]	Yin X, Zhang Y, Zhang L, Wang B, Zhao Y, Irfan M, Chen L, Feng Y. 2021. Regulation of MYB transcription factors of anthocyanin synthesis in lily flowers. Frontiers in Plant Science,12:761668.
[23]	Zhang J, Zhao J, Tan Q, Qiu X, Mei S. 2021. Comparative transcriptome analysis reveals key genes associated with pigmentation in radish(Raphanus sativus L.) skin and flesh. Scientific Reports, 11 (1):11434. doi: 10.1038/s41598-021-90633-5
[24]	Zhang Z, Chen C, Lin H, Jiang C Y, Zhao Y H, Guo Y S. 2025. The VvHY5-VvMYB24-VvMYBA 1 transcription factor cascade regulates the biosynthesis of anthocyanin in grape. Horticultural Plant Journal, 11 (3):1066-1077. doi: 10.1016/j.hpj.2024.02.005 URL
[25]	Zhou H, Lin-Wang K, Wang F, Espley R V, Ren F, Zhao J, Ogutu C, He H, Jiang Q, Allan A C, Han Y. 2019. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. New Phytologist, 221 (4):1919-1934. doi: 10.1111/nph.15486 pmid: 30222199
[26]	Zhou L J, Liu S H, Wang Y G, Wang Y X, Song A P, Jiang J F, Chen S M, Guan Z Y, Chen F D. 2024. CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium. Horticultural Plant Journal, 10 (1):194-204. doi: 10.1016/j.hpj.2023.02.011 URL
[27]	Zuo X, Miao C, Li M, Gu L, Yang X, Song C, Li M, Du J, Xie C, Liu X, Sun H, Li L, Zhang Z, Wang F. 2023. Purple Rehmannia:investigation of the activation of R2R3-MYB transcription factors involved in anthocyanin biosynthesis. Physiologia Plantarum, 175 (3):e13920. doi: 10.1111/ppl.v175.3 URL
[28]	Zuo X, Wang F Q, Li X R, Li M M. 2020. Transcriptome-based screening and the optimal reference genes for real-time quantitative PCR in Rehmannia chingii and R. henryi. Biologia Plantarum,64:798-806.

Functional Study of RcMYB4 in Regulating Anthocyanin Biosynthesis in Rehmannia chingii

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