Skip to main content

Advertisement

SpringerLink
Log in

Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum)

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

Abstract

microRNAs (miRNAs) are a recently discovered class of small (~21 nt) endogenous gene regulators that have been shown to play an important role in plant growth and development by aiding in organ maturation, hormone signaling, tissue differentiation, and plant tolerance to environmental stress. Since a list of miRNAs has never been generated for tobacco, we employed genome survey sequence analysis to computationally identify 259 potentially conserved tobacco miRNAs, belonging to 65 families, and validated 11 of these miRNAs using qRT-PCR. The 65 miRNA families were dramatically different in size. miRNA precursor (pre-miRNA) sequence analysis showed that tobacco pre-miRNAs greatly varied from 45 to 635 nt in length with an average of 141 ± 108 nt. We were also able to determine the presence of antisense miRNAs as well as miRNA clusters in tobacco. Using previously established protocols, a total of 1,225 potential target genes were predicted for the newly identified tobacco miRNAs. These target genes include transcription factors, DNA replication proteins, metabolic enzymes, as well as other gene targets necessary for proper plant maturation. The results of this study show that conserved miRNAs exist in tobacco and suggest that these miRNAs may play an important role in tobacco growth and development.

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
Fig. 7

Similar content being viewed by others

Abbreviations

AP2:

Apetala2-like

BLAST:

Basic local alignment search tool

CT :

Cycle threshold

EF1α:

Elongation factor 1α

EST:

Expressed sequence tag

GSS:

Genome survey sequence

MFE:

Minimal folding free energy

MFEI:

Minimal folding free energy index

miRNAs:

MicroRNAs

NCBI:

National Center for Biotechnology Information

nt:

Nucleotide

pre-miRNA:

miRNA precursor

pri-miRNA:

Primary miRNA

qRT-PCR:

Quantitative real-time PCR

RACE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse transcription PCR

SPL:

Squamosa promoter binding protein-like

References

  • Achard P, Herr A, Baulcombe DC, Harberd NP (2004) Modulation of floral development by a gibberellin-regulated microRNA. Development 131:3357–3365

    Article  CAS  PubMed  Google Scholar 

  • Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355

    Article  CAS  PubMed  Google Scholar 

  • Andrianov V, Borisjuk N, Pogrebnyak N, Brinker A, Dixon J, Spitsin S, Flynn J, Matyszczuk P, Andryszak K, Laurelli M, Golovkin M, Koprowski H (2010) Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. Plant Biotechnol J 8:277–287

    Article  CAS  PubMed  Google Scholar 

  • Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741

    Article  CAS  PubMed  Google Scholar 

  • Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

    Article  CAS  PubMed  Google Scholar 

  • Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2008) GenBank. Nucleic Acids Res 36:D25–D30

    Article  CAS  PubMed  Google Scholar 

  • Bonnet E, Wuyts J, Rouze P, Van de Peer Y (2004) Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences. Bioinformatics 20:2911–2917

    Article  CAS  PubMed  Google Scholar 

  • Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of microRNA-target recognition. PLoS Biol 3:404–418

    Article  CAS  Google Scholar 

  • Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179

    Article  PubMed  Google Scholar 

  • Cui X, Xu SM, Mu DS, Yang ZM (2009) Genomic analysis of rice microRNA promoters and clusters. Gene 431:61–66

    Article  CAS  PubMed  Google Scholar 

  • Emery JF, Floyd SK, Alvarez J, Eshed Y, Hawker NP, Izhaki A, Baum SF, Bowman JL (2003) Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes. Curr Biol 13:1768–1774

    Article  CAS  PubMed  Google Scholar 

  • Gleave AP, Ampomah-Dwamena C, Berthold S, Dejnoprat S, Karunairetnam S, Nain B, Wang Y-Y, Crowhurst RN, MacDiarmid RM (2008) Identification and characterisation of primary microRNAs from apple (Malus domestica cv. Royal Gala) expressed sequence tags. Tree Genet Genomes 4:343–358

    Article  Google Scholar 

  • Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158

    Article  CAS  PubMed  Google Scholar 

  • Guo HS, Xie Q, Fei JF, Chua NH (2005) MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17:1376–1386

    Article  CAS  PubMed  Google Scholar 

  • Guo Q, Xiang AL, Yang Q, Yang ZM (2007) Bioinformatic identification of microRNAs and their target genes from Solanum tuberosum expressed sequence tags. Chin Sci Bull 52:2380–2389

    Article  CAS  Google Scholar 

  • Han Y, Zhu B, Luan F, Zhu H, Shao Y, Chen A, Lu C, Luo Y (2010) Conserved miRNAs and their targets identified in lettuce (Lactuca) by EST analysis. Gene 463:1–7

    Article  CAS  PubMed  Google Scholar 

  • Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787–799

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20

    Article  CAS  PubMed  Google Scholar 

  • Li B, Yin W, Xia X (2009) Identification of microRNAs and their targets from Populus euphratica. Biochem Biophys Res Comm 388:272–277

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Fu Y, Ji L, Wu C, Zheng C (2010) Characterization and expression analysis of the Arabidopsis mir169 family. Plant Sci 178:271–280

    Article  CAS  Google Scholar 

  • Liu D, Song Y, Chen Z, Yu D (2009) Ectopic expression of miR396 suppresses GRF target gene expression and alters leaf growth in Arabidopsis. Physiol Plant 136:223–236

    Article  CAS  PubMed  Google Scholar 

  • Lu YZ, Yan DW, Lu YT (2005) Identification of microRNAs from rice. Funct Plant Biol 32:963–971

    Article  CAS  Google Scholar 

  • Mathews DH, Sabina J, Zuker M, Turner DH (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940

    Article  CAS  PubMed  Google Scholar 

  • Merchan F, Boualem A, Crespi M, Frugier F (2009) Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins. Genome Biol 10:R136

    Article  PubMed  Google Scholar 

  • Palatnik JF, Allen E, Wu XL, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263

    Article  CAS  PubMed  Google Scholar 

  • Park W, Li JJ, Song RT, Messing J, Chen XM (2002) CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12:1484–1495

    Article  CAS  PubMed  Google Scholar 

  • Qiu CX, Xie FL, Zhu YY, Guo K, Huang SQ, Nie L, Yang ZM (2007) Computational identification of microRNAs and their targets in Gossypium hirsutum expressed sequence tags. Gene 395:49–61

    Article  CAS  PubMed  Google Scholar 

  • Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626

    Article  CAS  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 

  • Song C, Fang J, Li X, Liu H, Chao CT (2009) Identification and characterization of 27 conserved microRNAs in citrus. Planta 230:671–685

    Article  CAS  PubMed  Google Scholar 

  • Stark A, Bushati N, Jan CH, Kheradpour P, Hodges E, Brennecke J, Bartel DP, Cohen SM, Kellis M (2008) A single Hox locus in Drosophila produces functional microRNAs from opposite DNA strands. Genes Dev 22:8–13

    Article  CAS  PubMed  Google Scholar 

  • Sunkar R (2010) MicroRNAs with macro-effects on plant stress responses. Stem Cell Dev Biol. doi:10.1016/j.semcdb.2010.04.001

  • Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of microRNAs from rice. Plant Cell 17:1397–1411

    Article  CAS  PubMed  Google Scholar 

  • Sunkar R, Kapoor A, Zhu JK (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065

    Article  CAS  PubMed  Google Scholar 

  • Talmor-Neiman M, Stav R, Frank W, Voss B, Arazi T (2006) Novel micro-RNAs and intermediates of micro-RNA biogenesis from moss. Plant J 47:25–37

    Article  CAS  PubMed  Google Scholar 

  • Unver T, Budak H (2009) Conserved microRNAs and their targets in model grass species Brachypodium distachyon. Planta 230:659–669

    Article  CAS  PubMed  Google Scholar 

  • Williams L, Grigg SP, Xie MT, Christensen S, Fletcher JC (2005) Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166 g and its AtHD-ZIP target genes. Dev 132:3657–3668

    Article  CAS  Google Scholar 

  • Wu G, Poethig RS (2006) Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133:3539–3547

    Article  CAS  PubMed  Google Scholar 

  • Xie FL, Huang SQ, Guo K, Xiang AL, Zhu YY, Nie L, Yang ZM (2007) Computational identification of novel microRNAs and targets in Brassica napus. FEBS Lett 581:1464–1474

    Article  CAS  PubMed  Google Scholar 

  • Xie FL, Frazier TP, Zhang B (2010) Identification and characterization of microRNAs and their targets in the bioenergy plant switchgrass (Panicum virgatum). Planta 232:417–434

    Article  CAS  PubMed  Google Scholar 

  • Yin Z, Shen F (2010) Identification and characterization of conserved microRNAs and their target genes in wheat (Triticum aestivum). Genet Mol Res 9:1186–1196

    Article  CAS  PubMed  Google Scholar 

  • Yin Z, Li C, Han X, Shen F (2008) Identification of conserved microRNAs and their target genes in tomato (Lycopersicon esculentum). Gene 414:60–66

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Wang QL, Cobb GP, Anderson TA (2005) Identification and characterization of new plant microRNAs using EST analysis. Cell Res 15:336–360

    Article  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Cannon CH, Cobb GP, Anderson TA (2006a) Conservation and divergence of plant microRNA genes. Plant J 46:243–259

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Anderson TA (2006b) Identification of 188 conserved maize microRNAs and their targets. FEBS Lett 580:3753–3762

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Cox SB, Cobb GP, Anderson TA (2006c) Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci 63:246–254

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Cobb GP, Anderson TA (2006d) Plant microRNA: a small regulatory molecule with big impact. Dev Biol 289:3–16

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Wang QL, Cobb GP, Anderson TA (2006e) Computational identification of microRNAs and their targets. Comput Biol Chem 30:395–407

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Wang QL, Wang KB, Pan XP, Liu F, Guo TL, Cobb GP, Anderson TA (2007) Identification of cotton microRNAs and their targets. Gene 397:26–37

    Article  CAS  PubMed  Google Scholar 

  • Zhang BH, Pan XP, Stellwag EJ (2008a) Identification of soybean microRNAs and their targets. Planta 229:161–182

    Article  CAS  PubMed  Google Scholar 

  • Zhang J, Zeng R, Chen J, Liu X, Liao Q (2008b) Identification of conserved microRNAs and their targets from Solanum lycopersicum Mill. Gene 423:1–7

    Article  CAS  PubMed  Google Scholar 

  • Zhang WW, Luo YP, Gong X, Zeng WH, Li SG (2009) Computational identification of 48 potato microRNAs and their targets. Comput Biol Chem 33:84–93

    Article  CAS  PubMed  Google Scholar 

  • Zhao B, Ge L, Liang R, Li W, Ruan K, Lin H, Jin Y (2009) Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Mol Biol 10:29

    Article  PubMed  Google Scholar 

  • Zhou XF, Sunkar R, Jin HL, Zhu JK, Zhang WX (2009) Genome-wide identification and analysis of small RNAs originated from natural antisense transcripts in Oryza sativa. Genome Res 19:70–78

    Article  CAS  PubMed  Google Scholar 

  • Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This project was partially supported by East Carolina University New Faculty Research Startup Funds Program and a Science and Engineering Grant from DuPont. We would like to thank Michael Bossé and Cornelius Flowers for their time spent on this project. We would also like to thank Dr. Ramsey Lewis and Mr. Ted Woodlief at North Carolina State University for kindly providing tobacco seeds. Finally, we want to thank Ms. Julie Marik of the East Carolina University Greenhouse Facility for her care of the tobacco plants.

Author information

Authors and Affiliations

  1. Department of Biology, East Carolina University, Greenville, NC, 27858, USA

    Taylor P. Frazier, Fuliang Xie, Andrew Freistaedter, Caitlin E. Burklew & Baohong Zhang

Authors
  1. Taylor P. Frazier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fuliang Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew Freistaedter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Caitlin E. Burklew
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Baohong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Baohong Zhang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1448 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frazier, T.P., Xie, F., Freistaedter, A. et al. Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum). Planta 232, 1289–1308 (2010). https://doi.org/10.1007/s00425-010-1255-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-010-1255-1

Keywords

Search

Navigation