Skip to main content
SpringerLink
Log in

Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Main conclusion

Our work strongly suggests that microRNA858 regulates anthocyanin biosynthesis in tomato by modulating the expression of two R2R3 MYB transcription factors, underscoring the importance of microRNAs in the gene regulatory network controlling specialized metabolism in plants.

The biological functions of microRNA858 (miR858), a recently identified small RNA, are not well understood. Here, we identified miR858 as a negative regulator of anthocyanin biosynthesis in tomato (Solanum lycopersicum). RNA ligase-mediated 5′RACE cleavage assay showed that miR858 mediates the cleavage of SlMYB7-like and SlMYB48-like transcripts in tomato. Expression analysis revealed an inverse correlation between the accumulation of miR858 and its target SlMYB7-like mRNA, in different tissues of tomato. Subsequently, a small tandem target mimic construct for blocking miR858 (STTM858) was generated and transformed into tomato. The majority of endogenous miR858 was blocked in STTM858 over-expressing tomato plants, whereas SlMYB7-like transcripts increased significantly. Concomitantly, upregulated expression was detected for several anthocyanin biosynthetic genes, including PAL, CHS, DFR, ANS and 3GT. As a result, anthocyanins were highly accumulated in young seedlings, leaves, stems and leaf buds of the transgenic plants under normal growth conditions. In addition, over-expression of STTM858 in tomato activated another MYB transcription factor, SlMYB48, implicating the possible involvement of SlMYB48 in anthocyanin biosynthesis.

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

Similar content being viewed by others

Abbreviations

ANS:

Anthocyanidin synthase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol reductase

miRNA:

MicroRNA

PAL:

Phenylalanine ammonialyase

pri-miRNA:

Primary miRNA

qRT-PCR:

Quantitative real-time reverse transcription PCR

STTM:

Small tandem target mimic

3GT:

Flavonol-3-glucosyltransferase

References

  • Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ (2008) Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 18:758–762

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ballester AR, Molthoff J, de Vos R, Hekkert B, Orzaez D, Fernandez-Moreno JP, Tripodi P, Grandillo S, Martin C, Heldens J, Ykema M, Granell A, Bovy A (2010) Biochemical and 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 Central  PubMed  Google Scholar 

  • Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

    Article  CAS  PubMed  Google Scholar 

  • Buer CS, Imin N, Djordjevic MA (2010) Flavonoids: new roles for old molecules. J Integr Plant Biol 52:98–111

    Article  CAS  PubMed  Google Scholar 

  • Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EG, Hall RD, Bovy AG, Luo J, Martin C (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26:1301–1308

    Article  CAS  PubMed  Google Scholar 

  • Castellarin SD, Pfeiffer A, Sivilotti P, Degan M, Peterlunger E, Dig G (2007) Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit. Plant, Cell Environ 30:1381–1399

    Article  CAS  Google Scholar 

  • Christie P, Alfenito M, Walbot V (1994) Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194:541–549

    Article  CAS  Google Scholar 

  • Distelfeld A, Pearce SP, Avni R, Scherer B, Uauy C, Piston F, Slade A, Zhao R, Dubcovsky J (2012) Divergent functions of orthologous NAC transcription factors in wheat and rice. Plant Mol Biol 78:515–524

    Article  CAS  PubMed  Google Scholar 

  • Dubos C, Le Gourrierec J, Baudry A, Huep G, Lanet E, Debeaujon I, Routaboul JM, Alboresi A, Weisshaar B, Lepiniec L (2008) MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J 55:940–953

    Article  CAS  PubMed  Google Scholar 

  • Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, Grant SR, Dangl JL, Carrington JC (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2:e219

    Article  PubMed Central  PubMed  Google Scholar 

  • Fahlgren N, Jogdeo S, Kasschau KD, Sullivan CM, Chapman EJ, Laubinger S, Smith LM, Dasenko M, Givan SA, Weigel D, Carrington JC (2010) MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana. Plant Cell 22:1074–1089

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fornale S, Lopez E, Salazar-Henao JE, Fernandez-Nohales P, Rigau J, Caparros-Ruiz D (2014) AtMYB7, a new player in the regulation of UV-sunscreens in Arabidopsis thaliana. Plant Cell Physiol 55:507–516

    Article  CAS  PubMed  Google Scholar 

  • Garcia-Mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G, Gonzalez VM, Henaff E, Camara F, Cozzuto L, Lowy E, Alioto T, Capella-Gutierrez S, Blanca J, Canizares J, Ziarsolo P, Gonzalez-Ibeas D, Rodriguez-Moreno L, Droege M, Du L, Alvarez-Tejado M, Lorente-Galdos B, Mele M, Yang L, Weng Y, Navarro A, Marques-Bonet T, Aranda MA, Nuez F, Pico B, Gabaldon T, Roma G, Guigo R, Casacuberta JM, Arus P, Puigdomenech P (2012) The genome of melon (Cucumis melo L.). Proc Natl Acad Sci USA 109:11872–11877

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gou JY, Felippes FF, Liu CJ, Weigel D, Wang JW (2011) Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23:1512–1522

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Guan X, Pang M, Nah G, Shi X, Ye W, Stelly DM, Chen ZJ (2014) miR828 and miR858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development. Nat Commun 5:3050

    Article  PubMed  Google Scholar 

  • He J, Silva AM, Mateus N, de Freitas V (2011) Oxidative formation and structural characterisation of new alpha-pyranone (lactone) compounds of non-oxonium nature originated from fruit anthocyanins. Food Chem 127:984–992

    Article  CAS  PubMed  Google Scholar 

  • Hsieh LC, Lin SI, Shih AC, Chen JW, Lin WY, Tseng CY, Li WH, Chiou TJ (2009) Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing. Plant Physiol 151:2120–2132

    Article  PubMed Central  PubMed  Google Scholar 

  • Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

    Article  CAS  PubMed  Google Scholar 

  • Li YY, Mao K, Zhao C, Zhao XY, Zhang HL, Shu HR, Hao YJ (2012) MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiol 160:1011–1022

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Lorenc-Kukula K, Jafra S, Oszmianski J, Szopa J (2005) Ectopic expression of anthocyanin 5-o-glucosyltransferase in potato tuber causes increased resistance to bacteria. J Agric Food Chem 53:272–281

    Article  CAS  PubMed  Google Scholar 

  • Luo QJ, Mittal A, Jia F, Rock CD (2012) An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in Arabidopsis. Plant Mol Biol 80:117–129

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Maier A, Schrader A, Kokkelink L, Falke C, Welter B, Iniesto E, Rubio V, Uhrig JF, Hulskamp M, Hoecker U (2013) Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis. Plant J 74:638–651

    Article  CAS  PubMed  Google Scholar 

  • Mathews H, Clendennen SK, Caldwell CG, Liu XL, Connors K, Matheis N, Schuster DK, Menasco DJ, Wagoner W, Lightner J, Wagner DR (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15:1689–1703

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Matsui K, Umemura Y, Ohme-Takagi M (2008) AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant J 55:954–967

    Article  CAS  PubMed  Google Scholar 

  • Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJ (2009) RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 21:367–376

    Article  CAS  PubMed  Google Scholar 

  • Mehrtens F, Kranz H, Bednarek P, Weisshaar B (2005) The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis. Plant Physiol 138:1083–1096

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Meiers S, Kemeny M, Weyand U, Gastpar R, von Angerer E, Marko D (2001) The anthocyanidins cyanidin and delphinidin are potent inhibitors of the epidermal growth-factor receptor. J Agric Food Chem 49:958–962

    Article  CAS  PubMed  Google Scholar 

  • Mooney M, Desnos T, Harrison K, Jones J, Carpenter R, Coen E (1995) Altered regulation of tomato and tobacco pigmentation genes caused by the delila gene of Antirrhinum. Plant J 7:333–339

    Article  CAS  Google Scholar 

  • Morishita T, Kojima Y, Maruta T, Nishizawa-Yokoi A, Yabuta Y, Shigeoka S (2009) Arabidopsis NAC transcription factor, ANAC078, regulates flavonoid biosynthesis under high-light. Plant Cell Physiol 50:2210–2222

    Article  CAS  PubMed  Google Scholar 

  • Moxon S, Jing R, Szittya G, Schwach F, Rusholme Pilcher RL, Moulton V, Dalmay T (2008) Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Res 18:1602–1609

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Oh S, Park S, Han KH (2003) Transcriptional regulation of secondary growth in Arabidopsis thaliana. J Exp Bot 54:2709–2722

    Article  CAS  PubMed  Google Scholar 

  • Patra B, Pattanaik S, Yuan L (2013a) Proteolytic degradation of the flavonoid regulators, TRANSPARENT TESTA8 and TRANSPARENT TESTA GLABRA1, in Arabidopsis is mediated by the ubiquitin/26Sproteasome system. Plant Signal Behav. doi:10.4161/psb25901

    PubMed Central  PubMed  Google Scholar 

  • Patra B, Pattanaik S, Yuan L (2013b) Ubiquitin protein ligase 3 mediates the proteasomal degradation of GLABROUS 3 and ENHANCER OF GLABROUS 3, regulators of trichome development and flavonoid biosynthesis in Arabidopsis. Plant J 74:435–447

    Article  CAS  PubMed  Google Scholar 

  • Patra B, Schluttenhofer C, Wu Y, Pattanaik S, Yuan L (2013c) Transcriptional regulation of secondary metabolite biosynthesis in plants. Biochim Biophys Acta 1829:1236–1247

    Article  CAS  PubMed  Google Scholar 

  • Qi T, Song S, Ren Q, Wu D, Huang H, Chen Y, Fan M, Peng W, Ren C, Xie D (2011) The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate Jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Plant Cell 23:1795–1814

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520

    Article  CAS  PubMed  Google Scholar 

  • Sarma AD, Sharma R (1999) Anthocyanin-DNA copigmentation complex: mutual protection against oxidative damage. Phytochemistry 52:1313–1318

    Article  CAS  Google Scholar 

  • Slavikova S, Ufaz S, Avin-Wittenberg T, Levanony H, Galili G (2008) An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses. J Exp Bot 59:4029–4043

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smith CJ, Watson CF, Bird CR, Ray J, Schuch W, Grierson D (1990) Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol Gen Genet 224:477–481

    Article  CAS  PubMed  Google Scholar 

  • Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456

    Article  CAS  PubMed  Google Scholar 

  • Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tanaka Y, Ohmiya A (2008) Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways. Curr Opin Biotechnol 19:190–197

    Article  CAS  PubMed  Google Scholar 

  • Tang G, Yan J, Gu Y, Qiao M, Fan R, Mao Y, Tang X (2012) Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods 58:118–125

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tominaga-Wada R, Nukumizu Y, Wada T (2013) Tomato (Solanum lycopersicum) homologs of TRIPTYCHON (SlTRY) and GLABRA3 (SlGL3) are involved in anthocyanin accumulation. Plant Signal Behav 8:e24575

    Article  PubMed Central  PubMed  Google Scholar 

  • Xia R, Zhu H, An YQ, Beers EP, Liu Z (2012) Apple miRNAs and tasiRNAs with novel regulatory networks. Genome Biol 13:R47

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Yang F, Cai J, Yang Y, Liu Z (2013) Overexpression of microRNA828 reduces anthocyanin accumulation in Arabidopsis. Plant Cell Tissue Organ Cult (PCTOC) 115:159–167

    Article  CAS  Google Scholar 

  • Zhu H, Xia R, Zhao B, An YQ, Dardick CD, Callahan AM, Liu Z (2012) Unique expression, processing regulation, and regulatory network of peach (Prunus persica) miRNAs. BMC Plant Biol 12:149

    Article  CAS  PubMed Central  PubMed  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 

  • Zuluaga D, Gonzali S, Loreti E, Pucciariello C, Innocenti E, Guidi L, Alpi A, Perata P (2008) Arabidopsis thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants. Funct Plant Biol 35:606–618

    Article  CAS  Google Scholar 

Download references

Acknowledgments

X. J. is supported by NSFC (31101555), NSF of Shanxi Province (2011021032-2), Program for Leading Young Scientists of Shanxi High Education (2012), Returned Overseas Scholar Research Fund of Shanxi (2011051), Talent Introduction and Development of Shanxi (614191), Science and Technology Innovation Fund of Shanxi Agricultural University (2010021). Research in L. Y. lab is partially supported by a grant from the United States National Science Foundation (Award number 1355438) and the Kentucky Tobacco Research and Development Center.

Author information

Authors and Affiliations

  1. College of Life Science, Shanxi Agricultural University, Taigu, 030801, Shanxi, China

    Xiaoyun Jia, Jie Shen, Hui Liu, Fang Li, Na Ding, Changyong Gao, Runzhi Li & Ling Yuan

  2. Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu, 030801, China

    Ling Yuan

  3. Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, 40546, USA

    Sitakanta Pattanaik, Barunava Patra & Ling Yuan

Authors
  1. Xiaoyun Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jie Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Na Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Changyong Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sitakanta Pattanaik
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Barunava Patra
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Runzhi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ling Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Runzhi Li or Ling Yuan.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jia, X., Shen, J., Liu, H. et al. Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato. Planta 242, 283–293 (2015). https://doi.org/10.1007/s00425-015-2305-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-015-2305-5

Keywords

Search

Navigation