Skip to main content
SpringerLink
Log in

IbERF71, with IbMYB340 and IbbHLH2, coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potato (Ipomoea batatas L.)

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

The transcription factor (TF) IbERF71 forms a novel complex, IbERF71-IbMYB340-IbbHLH2, to coregulate anthocyanin biosynthesis by binding to the IbANS1 promoter in purple-fleshed sweet potatoes.

Abstract

Purple-fleshed sweet potato (Ipomoea batatas L.) is very popular because of its abundant anthocyanins, which are natural pigments with multiple physiological functions. TFs involved in regulating anthocyanin biosynthesis have been identified in many plants. However, the molecular mechanism of anthocyanin biosynthesis in purple-fleshed sweet potatoes has rarely been examined. In this study, TF IbERF71 and its partners were screened by bioinformatics and RT-qPCR analysis. The results showed that the expression levels of IbERF71 and partners IbMYB340 and IbbHLH2 were higher in purple-fleshed sweet potatoes than in other colors and that the expression levels positively correlated with anthocyanin contents. Moreover, transient expression assays showed that cotransformation of IbMYB340+IbbHLH2 resulted in anthocyanin accumulation in tobacco leaves and strawberry receptacles, and additional IbERF71 significantly increased visual aspects. Furthermore, the combination of the three TFs significantly increased the expression levels of FvANS and FvGST, which are involved in anthocyanin biosynthesis and transport of strawberry receptacles. The dual-luciferase reporter system verified that cotransformation of the three TFs enhanced the transcription activity of IbANS1. In addition, yeast two-hybrid and firefly luciferase complementation assays revealed that IbMYB340 interacted with IbbHLH2 and IbERF71 but IbERF71 could not interact with IbbHLH2 in vitro. In summary, our findings provide novel evidence that IbERF71 and IbMYB340-IbbHLH2 form the regulatory complex IbERF71-IbMYB340-IbbHLH2 that coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Thus, the present study provides a new regulatory network of anthocyanin biosynthesis and strong insight into the color development of purple-fleshed sweet potatoes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3.
Fig. 4.
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • An JP, Wang XF, Li YY, Song LQ, Zhao LL, You CX, Hao YJ (2018) EIN3-LIKE1, MYB1, and ETHYLENE RESPONSE FACTOR3 act in a regulatory loop that synergistically modulates ethylene biosynthesis and anthocyanin accumulation. Plant Physiol. 178(2):808–823

    CAS  PubMed  PubMed Central  Google Scholar 

  • Castellarin SD, Di Gaspero G (2007) Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines. BMC Plant Biol. 7:46

    PubMed  PubMed Central  Google Scholar 

  • Chagné D, Lin-Wang K, Espley RV et al (2013) An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes. Plant Physiol. 161(1):225–239

    PubMed  Google Scholar 

  • Choi JH, Hwang YP, Choi CY, Chung YC, Jeong HG (2010) Anti-fibrotic effects of the anthocyanins isolated from the purple-fleshed sweet potato on hepatic fibrosis induced by dimethylnitrosamine administration in rats. Food Chem. Toxicol. 48(11):3137–3143

    CAS  PubMed  Google Scholar 

  • Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 53(5):814–827

    CAS  PubMed  Google Scholar 

  • Han Z, Hu Y, Lv Y, Rose JKC, Sun Y, Shen F, Wang Y, Zhang X, Xu X, Wu T, Han Z (2008) Natural variation underlies differences in ETHYLENE RESPONSE FACTOR 17 activity in fruit peel degreening. Plant Physiol. 176(3):2292–2304

    Google Scholar 

  • Hawkins C, Caruana J, Schiksnis E, Liu Z (2016) Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry. Sci. Rep. 6:29017

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA (2005) Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13

    PubMed  PubMed Central  Google Scholar 

  • Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7(7):1071–1083

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hwang YP, Choi JH, Yun HJ, Han EH, Kim HG, Kim JY, Park BH, Khanal T, Choi JM, Chung YC, Jeong HG (2011) Anthocyanins from purple-fleshed sweet potato attenuate dimethylnitrosamine-induced liver injury in rats by inducing Nrf2-mediated antioxidant enzymes and reducing COX-2 and iNOS expression. Food Chem. Toxicol. 49(1):93–99

    CAS  PubMed  Google Scholar 

  • Jaakola L, Poole M, Jones MO et al (2010) A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant Physiol. 153(4):1619–1629

    CAS  PubMed  PubMed Central  Google Scholar 

  • Julien P, Fabiola JM, Teresa SBM et al (2006) Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination. Plant Cell Physiol. 47(9):1195–1205

    Google Scholar 

  • Kim CY, Ahn YO, Kim SH, Kim YH, Lee HS, Catanach AS, Jacobs JM, Conner AJ, Kwak SS (2010) The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system. Physiol Plant. 139(3):229–240

    CAS  PubMed  Google Scholar 

  • Kitamura S, Shikazono N, Tanaka A (2004) TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J. 37(1):104–114

    CAS  PubMed  Google Scholar 

  • Kou M, Liu YJ, Li ZY, Zhang YG, Tang W, Yan H, Wang X, Chen XG, Su ZX, Mohamed HA, Li Q, Ma DF (2019) A novel glutathione S-transferase gene from sweet potato, IbGSTF4, is involved in anthocyanin sequestration. Plant Physiol. Biochem. 135:395–403

    CAS  PubMed  Google Scholar 

  • Kwak SS (2019) Biotechnology of the sweetpotato: ensuring global food and nutrition security in the face of climate change. Plant Cell Rep. 38(11):1361–1363

    CAS  PubMed  Google Scholar 

  • Li C, Wu J, Hu KD, Wei SW, Sun HY, Hu LY, Han Z, Yao GF, Zhang H (2020) PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears. Hortic. Res. 7:37

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lin-Wang K, Micheletti D, Palmer J, Volz R, Lozano L, Espley R, Hellens RP, Chagnè D, Rowan DD, Troggio M, Iglesias I, Allan AC (2011) High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. Plant Cell Environ. 34(7):1176–1190

    PubMed  Google Scholar 

  • Liu XF, Yin XR, Andrew C, Wang KL, Shi YN, Huang YJ, Ian B, Xu CJ, Chen KS (2013) The role of MrbHLH1 and MrMYB1 in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra) during anthocyanin biosynthesis. Plant Cell Tiss Org. 115(3):285–298

    CAS  Google Scholar 

  • Liu Y, Jiang H, Zhao Y, Li X, Dai X, Zhuang J, Zhu M, Jiang X, Wang P, Gao L, Xia T (2019) Three Camellia sinensis glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanidins. Planta 250(4):1163–1175

    CAS  PubMed  Google Scholar 

  • Liu YH, Lin-Wang K, Espley Richard V, Wang L, Yang HY, Yu B, Dare A, Varkonyi-Gasic E, Wang J, Zhang JL, Wang D, Allan AC (2016) Functional diversification of the potato R2R3 MYB anthocyanin activators AN1, MYBA1, and MYB113 and their interaction with basic helix-loop-helix cofactors. J. Exp Bot. 67(8):2159–2176

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Y, Lin-Wang K, Deng C, Warran B, Wang L, Yu B, Yang H, Wang J, Espley RV, Zhang J, Wang D, Allan AC (2015) Comparative transcriptome analysis of white and purple potato to identify genes involved in anthocyanin biosynthesis. Plos One 10(6):e0129148

    PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408

    CAS  PubMed  Google Scholar 

  • Luo H, Dai C, Li Y, Feng J, Liu Z1, Kang C, (2018) RAP codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry. J. Exp Bot. 69(10):2595–2608

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mano H, Ogasawara F, Sato K, Higo H, Minobe Y (2007) Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato. Plant Physiol. 143(3):1252–1268

    CAS  PubMed  PubMed Central  Google Scholar 

  • Medina-Puche L, Cumplido-Laso G, Amil-Ruiz F, Hoffmann T, Ring L, Rodríguez-Franco A, Caballero JL, Schwab W, Muñoz-Blanco J, Blanco-Portales R (2014) MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of Fragaria×ananassa fruits. J. Exp Bot. 65(2):401–417

    CAS  PubMed  Google Scholar 

  • Moyano E, Martínez-Garcia JF, Martin C (1996) Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in antirrhinum flowers. Plant Cell 8(9):1519–1532

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 140(2):411–432

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ni J, Bai S, Zhao Y, Qian M, Tao R, Yin L, Gao L, Teng Y (2019) Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in ‘RedZaosu’ pear fruits by interacting with MYB114. Plant Mol. Biol. 99(1–2):67–78

    CAS  PubMed  Google Scholar 

  • Riechmann JL, Heard J, Martin G et al (2000) Arabidopsis Transcription factors: genome-wide comparative analysis among eukaryotes. Science 290(5499):2105–2110

    CAS  Google Scholar 

  • Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-Binding Specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem. Bioph. Res. Co. 290(3):998–1009

    CAS  Google Scholar 

  • Schaart JG, Dubos C, Romero De La Fuente I, van Houwelingen AM, de Vos RC, Jonker HH, Xu W, Routaboul JM, Lepiniec L, Bovy AG (2013) Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria × ananassa) fruits. New Phytol. 197(2):454–467

    CAS  PubMed  Google Scholar 

  • Sewelam N, Kazan K, Thomas-Hall SR, Kidd BN, Manners JM, Schenk PM (2013) Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis. Plos One 8(8):e70289

    CAS  PubMed  PubMed Central  Google Scholar 

  • Slovin JP, Schmitt K, Folta KM. (2009) An inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae. Plant Methods 5(1):15.

  • Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1(4):2019–2025

    CAS  PubMed  Google Scholar 

  • Singer SD, Liu Z, Cox KD (2012) Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators. Plant Cell Rep. 31(1):13–25

    CAS  PubMed  Google Scholar 

  • Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) Retracted: an enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33:949–956

    CAS  PubMed  Google Scholar 

  • Wei ZZ, Hu KD, Zhao DL, Tang J, Huang ZQ, Jin P, Li YH, Han Z, Hu LY, Yao GF、Zhang H, (2020) MYB44 competitively inhibits the formation of the MYB340-bHLH2-NAC56 complex to regulate anthocyanin biosynthesis in purple-fleshed sweet potato. BMC Plant Biol. 20:258

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xu W, Dubos C, Lepiniec L (2015) Transcriptional control of flavonoid biosynthesis by MYB–bHLH–WDR complexes. Trends Plant Sci. 20(3):176–185

    CAS  PubMed  Google Scholar 

  • Xu ZS, Yang QQ, Feng K, Xiong AS (2019) Changing Carrot Color: Insertions in DcMYB7 Alter the Regulation of Anthocyanin Biosynthesis and Modification. Plant Physiol. 181(1):195–207

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yang YF, Shi DY, Wang YN, Zhang L, Chen XG, Yang XP, Xiong HZ, Gehendra B, Waltram R, Dotun O, Yang GH, Shi AN (2020) Transcript profiling for regulation of sweet potato skin color in Sushu8 and its mutant Zhengshu20. Plant Physiol. Biochem. 148:1–9

    PubMed  Google Scholar 

  • Yao G, Ming M, Allan AC, Gu C, Li L, Wu X, Wang R, Chang Y, Qi K, Zhang S, Wu J (2017) Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. Plant J 92(3):437–451

    CAS  PubMed  Google Scholar 

  • Yin XR, Allan AC, Chen KS, Ferguson IB (2010) Kiwifruit EIL and ERF Genes Involved in Regulating Fruit Ripening. Plant Physiol. 153(3):1280–1292

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang J, Xu H, Wang N, Jiang S, Fang H, Zhang Z, Yang G, Wang Y, Su M, Xu L, Chen X (2018) The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple. Plant Mol. Biol. 98(3):205–218

    CAS  PubMed  Google Scholar 

  • Zhang ZF, Lu J, Zheng YL, Wu DM, Hu B, Shan Q, Cheng W, Li MQ, Sun YY (2013) Purple-fleshed sweet potato color attenuates hepatic insulin resistance via blocking oxidative stress and endoplasmic reticulum stress in high-fat-diet-treated mice. J. Nutr. Biochem. 24(6):1008–1018

    CAS  PubMed  Google Scholar 

  • Zhang JX, Lei YY, Wang BT, Li S, Yu S, Wang Y, Li H, Liu YX, Ma Y, Dai HY, Wang JH, Zhang ZH (2020) The high-quality genome of diploid strawberry (Fragaria nilgerrensis) provides new insights into anthocyanin accumulation. Plant Biotechnol. J. https://doi.org/10.1111/pbi.13351.

  • Zhang K, Wu ZD, Li YH, Zhang H, Wang LP, Zhou QL, Tang DB, Fu YF, He FF, Jiang YC, Yang H, Wang JC (2014) ISSR-based molecular characterization of an elite germplasm collection of sweet potato (Ipomoea batatas L) in China. J Integr Agric 13(11):2346–2361

    Google Scholar 

  • Zhao QZ, Zhao SY, Xia GM (2005) Research advances on the mechanism of RNA silencing in plants. Acta Genet. Sin. 32(1):104–110

    CAS  PubMed  Google Scholar 

  • Zhao ZC, Hu GB, Hu FC, Wang HC, Yang ZY, Lai B (2012) The UDP glucose: flavonoid-3-O-glucosyltransferase (UFGT) gene regulates anthocyanin biosynthesis in litchi (Litchi chinesis Sonn) during fruit coloration. Mol Biol Rep 39(6):6409–6415

    CAS  PubMed  Google Scholar 

  • Zhou H, Lin-Wang K, Wang H, Gu C, Dare AP, Espley RV, He H, Allan AC, Han Y (2015) Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J. 82(1):105–121

    CAS  PubMed  Google Scholar 

  • Zhou ZY, Bi GZ, Zhou JM (2018) Luciferase complementation assay for protein-protein interactions in plants. Curr Protoc Plant Biol 3:42–50

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Earmarked Fund for the China Agriculture Research System (CARS-10-B1), the National Key R&D Program of China (2019YFD1001303, 2019YFD1001300), the National Natural Science Foundation of China (31901993, 31970312, 31970200, 31872078, 31670278), the Natural Science Foundations of Anhui Province (1908085MC72), the Key Research and Development Program of Anhui Province (201904a06020031), the Fundamental Research Funds for the Central Universities (JZ2020YYPY0249, JZ2018HGBZ0160), National Undergraduate Training Programs for Innovation of China (No.202010359064, S202010359221). We thank Dr. Andrew C. Allan, Dr. Lin-Wang Kui and Dr. Richard Espley for the dual-vector pGreen II 0800-LUC in The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.

Author information

Author notes

  1. Zhiyuan Ning, Kangdi Hu and Zhilin Zhou authors contributed equally to this work.

Authors and Affiliations

  1. School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China

    Zhiyuan Ning, Kangdi Hu, Lixia Li, Chen Ding, Xiaoyan Chen, Gaifang Yao & Hua Zhang

  2. Xuzhou Institute of Agricultural Sciences of the Xuhuai District of Jiangsu Province, Jiangsu Xuzhou Sweetpotato Research Center, Xuzhou, 221131, China

    Zhilin Zhou, Donglan Zhao & Jun Tang

  3. Institute of Pomology/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China

    Hong Wang

Authors
  1. Zhiyuan Ning
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kangdi Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhilin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Donglan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lixia Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chen Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiaoyan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gaifang Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hua Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.Z.Y., K.D.H., G.F.Y. and H.Z. conceived and designed the experiments; N.Z.Y., G.F.Y., Z.Z.L., Z.D.L. and D.C. performed the experiments; C.X.Y., L.L.X., W.H., Z.Z.L. and T.J. analyzed the data; N.Z.Y. and G.F.Y. wrote the paper; K.D.H., G.F.Y. and H.Z. interpreted the data and revised the manuscript

Corresponding authors

Correspondence to Gaifang Yao or Hua Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by Neal Stewart.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ning, Z., Hu, K., Zhou, Z. et al. IbERF71, with IbMYB340 and IbbHLH2, coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potato (Ipomoea batatas L.). Plant Cell Rep 40, 157–169 (2021). https://doi.org/10.1007/s00299-020-02621-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-020-02621-0

Keywords

Search

Navigation