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The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

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Abstract

Key message

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.

Abstract

Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

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Abbreviations

CHS:

Chalone synthase

FLS:

Flavonol synthase

DFR:

Dihydroflavonol reductase

C4H:

Cinnamate 4-hydroxylase

ANS:

Anthocyanin synthase

UFGT:

UDP-glucose:flavonoid 3- glucosyltransferase

EAR:

ERF-associated amphiphilic repression

GFP:

Green fluorescent protein

GST:

Glutathione S-transferase

His:

Histidine

Anti:

Antibody

MS:

Murashige and Skoog

-T(-Trp):

No tryptophan

-L(-Leu):

No leucine

-H(-His):

No histidine

-A(-Ade):

No adenine

AD:

Activation domain

BD:

Binding domain

CDS:

Coding DNA sequence

References

  • Aharoni A, De vos C, Wein M, Sun Z, Greco R, Kroon A, Mol JN, O’connell AP (2001) The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J 28(3):319–332

    Article  CAS  PubMed  Google Scholar 

  • Albert NW, Lewis DH, Zhang H, Schwinn KE, Jameson PE & Davies KM (2011) Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning. Plant J 65:771–784

    Article  CAS  PubMed  Google Scholar 

  • Albert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolis C, Boase MR, Ngo H, Jameson PE, Schwinn KE (2014) A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26:962–980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • An XH, Tian Y, Chen KQ, Liu XJ, Liu DD, Xie XB, Cheng CG, Cong PH, Hao YJ (2014) MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol 56(4):650–662

    Article  PubMed  Google Scholar 

  • Azuma A, Kobayashi S, Mitani N, Shiraishi M, Yamada M, Ueno T, Kono A, Yakushiji H, Koshita Y (2008) Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin. Theor Appl Genet 117:1009–1019

    Article  CAS  PubMed  Google Scholar 

  • Bai SL, Saito A, Honda C, Hatsuyama Y, Ito A, Moriguchi T (2014) An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis. Planta 240:1051–1062

    Article  CAS  PubMed  Google Scholar 

  • Ballester AR, Molthoff J, de Vos R, te Lintel HB, Orzaez D, Fernandezmoreno JP, Tripodi P, Grandillo S, Martin C, Heldens J, Ykema M, Granell A, Bovy A (2010) Biochemical molecular analysis of pink tomatoes, deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color. Plant Physiol 152:71–84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol 48:958–970

    Article  CAS  PubMed  Google Scholar 

  • Boyer J, Liu RH (2004) Apple phytochemicals and their health benefits. Nutr J 3:5

    Article  PubMed  PubMed Central  Google Scholar 

  • Cavallini E, Matus JT, Finezzo L, Zenoni S, Loyola R, Guzzo F, Schlechter R, Ageorges A, Arce-Johnson P, Tornielli GB (2015) The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine. Plant Physiol 167:1448–1470. doi:10.1104/pp.114.256172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen X, Feng T, Zhang Y, He T, Feng J, Zhang C (2007) Genetic diversity of volatile components in xinjiang wild apple (Malus sieversii). J Genetic Genom 34(2):171–179

    Article  CAS  Google Scholar 

  • Chen XS, Zhang J, Liu DL, Ji XH, Zhang ZY, Zhang R, Mao ZQ, Zhang YM, Wang LX, Li M. 2014. Genetic Variation of F1 population between Malus sieversii f. neidzwetzkyana and apple varieties and evaluation on fruit characters of functional apple excellent strains. Sci Agric Sin 47(11), 2193–2204

    CAS  Google Scholar 

  • Chikako H, Nobuhiro K, Masato W, Satoru K, Shozo K, Junichi S, Zilian Z, Tomomi T, Takaya M (2002) Anthocyanin biosynthetic gene are coordinately expressed during red coloration in apple skin. Plant Physiol Biochem 40:955–962

    Article  Google Scholar 

  • Ciftci-Yilmaz S, Morsy MR, Song LH, Coutu A, Krizek BA, Lewis MW, Warren D, Cushman J, Connolly EL, Mittler R (2007) The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress. J Biol Chem 282:9260–9268

    Article  CAS  PubMed  Google Scholar 

  • Colquhoun TA, Kim JY, Wedde AE, Levin LA, Schmitt KC, Schuurink RC, Clark DG (2011) PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H. J Exp Bot 62:1133–1143

    Article  CAS  PubMed  Google Scholar 

  • Cook NC, Samman S (1996) Flavonoids-Chemistry, metabolism, cardiop rotective effects, and dietary sources. J Nutr Biochem 7:66–76

    Article  CAS  Google Scholar 

  • Czemmel S, Stracke R, Weisshaar B, Cordon N, Harris NN, Walker AR, Robinson SP, Bogs J (2009) The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol fynthesis in feveloping Grape berries. Plant Physiol 151:1513–1530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor MdMYB10. Plant J 49:414–427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Espley RV, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill J, Schouten HJ, Gardiner SE, Hellens RP, Allan AC (2009) Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 21:168–183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Feng SQ, Wang YL, Song Y, Xu YT, Chen XS (2010) Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta 232:245–255

    Article  CAS  PubMed  Google Scholar 

  • Hemm MR, Herrmann KM, Chapple C (2001) AtMYB4:A transcription factor general in the battle against UV. Trends Plant Sci 6(4):135–136

    Article  CAS  PubMed  Google Scholar 

  • Hill K, Wang H, Perry SE. (2008). A transcriptional repression motif in the MADS factor AGL15 is involved in recruitment of histone deacetylase complex components. Plant J 53(1), 172–185

    Article  CAS  PubMed  Google Scholar 

  • Hiratsu K, Ohta M, Matsui K, Ohme-Takagi M (2002) The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers. FEBS Lett 514(2):351–354

    Article  CAS  PubMed  Google Scholar 

  • Hiratsu K, Kyoko M, Koyama T, Ohme-Takagi M (2003) Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J 34(5):733–739

    Article  CAS  PubMed  Google Scholar 

  • Hollman P, Katan M (1997) Absorption, metabolism and health effects of dietary flavonoids in man. Biomed Pharmacother 51:305–310

    Article  CAS  PubMed  Google Scholar 

  • Ji XH, Wang YT, Zhang R, Wu SJ, An MM, Li M, Wang CZ, Chen XL, Zhang YM, Chen XS (2015) Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f.niedzwetzkyana). Plant Cell Tiss Organ Cult 120:325–337

    Article  CAS  Google Scholar 

  • Jin HL, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J 19:6150–6161. doi:10.1093/emboj/19.22.6150

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jun JY, Liu CG, Xiao XR, Dixon RA (2015) The Transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula. Plant Cell. doi:10.1105/tpc.15.00476

    PubMed  PubMed Central  Google Scholar 

  • Kam J, Gresshoff PM, Shorter R, Xue GP (2008) The Q-type C2H2 zinc finger subfamily of transcription factors in Triticum aestivum is predominantly expressed in roots and enriched with members containing an EAR repressor motif and responsive to drought stress. Plant Mol Biol 67(3):305–322

    Article  CAS  PubMed  Google Scholar 

  • Kazan K (2006) Negative regulation of defence and stress genes by EAR-motif-containing repressors. Trends Plant Sci 11(3):109–112

    Article  CAS  PubMed  Google Scholar 

  • Kenneth JL, Thomas DS (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2—ΔΔCT method. Methods 25:402–408

    Article  Google Scholar 

  • Kim SY, Lee JR, Kim SR (2006) Characterization of an Apple anthocyanidin synthase gene in transgenic Tobacco. Plants. J Plant Biol 49(4):326–330

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Kondo S, Hiraoka K, Kobayashi S, Honda C, Terahara N (2002) Changes in the expression of anthocyanin biosynthetic genes during apple development. J Am Soc Hortic Sci 127:971–976

    CAS  Google Scholar 

  • Liu XF, Li F, Yin XR, Xu CJ, Chen KS (2013) Recent advances in the transcriptional regulation of anthocyanin biosynthesis. Acta Hortic Sinica 40(11):2295–2306

    Article  CAS  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

    Article  PubMed  Google Scholar 

  • Mitsuda N, Todaka D, Nakashima K, Yamaguchi-Shinozaki K, Ohme-Takagi M (2006) Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice. Plant Biotechnol J 4:325–332

    Article  CAS  PubMed  Google Scholar 

  • Nocker SV, Berry G, Najdowski J, Michelutti R, Luffman M, Forsline P, Alsmairat N, Beaudry R, Muraleedharan GN, Ordidge M (2012) Genetic diversity of red-fleshed apples (Malus). Euphytica 185:281–293

    Article  Google Scholar 

  • Ohta M, Matsui K, Shinshi H, Ohme-Takagi M (2001) Repression domains of class II ERF transcriptional repressors share an essential motif for active repression. Plant Cell 13:1959–1968

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Paolocci F, Robbins MP, Passeri V, Hauck B, Morris P, Rubini A, Arcioni S, Damiani F (2011) The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves. J Exp Bot 62(3):1189–1200

    Article  CAS  PubMed  Google Scholar 

  • Preston J, Wheeler J, Heazlewood J, Li SF, Parish RW. 2004. AtMYB32 is required for normal pollen development in Arabidopsis thaliana. Plant J 40(6), 979–995

    Article  CAS  PubMed  Google Scholar 

  • Schaefer HM, McGraw K, Catoni C (2008) Birds use fruit colour as honest signal of dietary antioxidant rewards. Funct Ecol 22(2):303–310

    Article  Google Scholar 

  • Solfanelli C, Bartolini S, Vitagliano C, Lorenzi R (2006) Immunolocalization and quantification of IAA after self- and freepollination in Olea europaea L. Sci Hortic 110(4):345–351

    Article  CAS  Google Scholar 

  • Sonbol FM, Fornale S, Capellades M, Encina A, Tourino S, Torres JS, Rovira P, Ruel K, Puigdomenech P, Rigau J, Caparros-Ruiz D (2009) The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana. Plant Mol Biol 70:283–296

    Article  CAS  PubMed  Google Scholar 

  • Sun CD, Huang HZ, Xu CJ, Li X, Chen KS (2013) Biological activities of extracts from Chinese bayberry(Myrica rubra Sieb. et Zucc): a review. Plant Foods Hum Nutr 68(2):97–106

    Article  CAS  PubMed  Google Scholar 

  • Takos AM, Jaffé FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol 142(3):1216–1232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tiwari SB, Wang XJ, Hagen G, Guilfoyle TJ (2001) Aux/IAA proteins are active repressors and their stability and activity are modulated by auxin. Plant Cell 13:2809–2822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tiwari SB, Hagen G, Guilfoyle TJ (2004) Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell 16:533–543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsukagoshi H, Morikami A, Nakamura K. 2007. Two B3 domain transcriptional repressors prevent sugar-inducible expression of seed maturation genes in Arabidopsis seedlings. Proc Nat Acad Sci USA 104(7), 2543–2547

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Velasco R et al (2010) The genome of the domesticated apple (Malus domestica Borkh). Nat Genet 42:833–839

    Article  CAS  PubMed  Google Scholar 

  • Wang LK, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK, Espley RV, Hellens RP, Allan AC (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol 10:50

    Article  CAS  Google Scholar 

  • Wang N, Zheng Y, Duan NB, Zhang ZY, Ji XH, Jiang SH, Sun SS, Yang L, Bai Y, Fei ZJ, Chen XS. (2015). Comparative Transcriptomes Analysis of Red and White-Fleshed Apples in an F1 Populationof Malus sieversii f. niedzwetzkyana Crossed with M. domestica ‘Fuji’. PLoS ONE. 10(7):e0133468

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu F, Ning Y, Zhang W, Liao Y, Li L, Cheng H, Cheng S (2014a) An R2R3-MYB transcription factor as a negative regulator of the flavonoid biosynthesis pathway in Ginkgo biloba. Funct Integr Genom 14(1):177–189

    Article  Google Scholar 

  • Xu W, Grain D, Bobet S, Le Gourrierec J, Thévenin J, Kelemen Z, Lepiniec L, Dubos C (2014b) Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-bHLHWDR complexes and their targets in Arabidopsis seed. New Phytol 202:132–144

    Article  CAS  PubMed  Google Scholar 

  • Zhu L, Shan H, Chen SM, Jiang JF, Gu CS, Zhou GQ, Chen Y, Song AP, Chen FD.2013. The Heterologous expression of the Chrysanthemum R2R3-MYB transcription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana. PLoS ONE 8(6), e65680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant J 40:22–34

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31572091) and National Key Research and Development Project of China (2016YFC0501505).

Author contributions

XC and HX: conceived and designed the experiments. HX and NW: performed the experiments. HX: analyzed the data. JL, CQ, YW, SJ, NL, ZZ: contributed reagents/materials/analysis tools. HX and XC: wrote the paper.

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Authors and Affiliations

  1. National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai-An, Shandong, China

    Haifeng Xu, Nan Wang, Jingxuan Liu, Changzhi Qu, Yicheng Wang, Shenghui Jiang, Deyun Wang, Zongying Zhang & Xuesen Chen

  2. Shandong institute of pomology, Tai-An, Shandong, China

    Ninglin Lu

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  1. Haifeng Xu
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  2. Nan Wang
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  3. Jingxuan Liu
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  9. Zongying Zhang
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  10. Xuesen Chen
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Correspondence to Xuesen Chen.

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Xu, H., Wang, N., Liu, J. et al. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol Biol 94, 149–165 (2017). https://doi.org/10.1007/s11103-017-0601-0

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