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Abiotic Stress-Associated miRNAs: Detection and Functional Analysis

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Plant MicroRNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 592))

Abstract

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.

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Notes

  1. 1.

    The number of cycles required may vary according to the organism or tissue.

  2. 2.

    The number of cycles required may vary according to the organism or tissue.

References

  1. 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 

  2. Chen X (2005) MicroRNA biogenesis and function in plants. FEBS Lett 579:5923–5931

    Article  CAS  PubMed  Google Scholar 

  3. Vazquez F (2006) Arabidopsis endogenous small RNAs: highways and byways. Trends Plant Sci 11:460–468

    Article  CAS  PubMed  Google Scholar 

  4. Park W, Li J, Song R, Messing J, Chen X (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 

  5. Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18:1187–1197

    Article  CAS  PubMed  Google Scholar 

  6. Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056

    Article  CAS  PubMed  Google Scholar 

  7. Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CL (2006) Regulation of phosphate homeostasis by MicroRNA in Arabidopsis. Plant Cell 18:412–421

    Article  CAS  PubMed  Google Scholar 

  8. 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 

  9. Zhang JF, Yuan LJ, Shao Y, Du W, Yan DW, Lu YT (2008) The disturbance of small RNA pathways enhanced abscisic acid response and multiple stress responses in Arabidopsis. Plant Cell Environ 31:562–574

    Article  CAS  PubMed  Google Scholar 

  10. 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 

  11. Zhou X, Wang G, Sutoh K, Zhu JK, Zhang W (2008) Identification of cold-inducible microRNAs in plants by transcriptome analysis. Biochim Biophys Acta 1779:780–788

    CAS  PubMed  Google Scholar 

  12. Zhou X, Wang G, Zhang W (2007) UV-B responsive microRNA genes in Arabidopsis thaliana. Mol Syst Biol 3:103

    Article  PubMed  Google Scholar 

  13. Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019

    Article  CAS  PubMed  Google Scholar 

  14. Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK (2005) A miRNA involved in phosphate-starvation response in Arabidopsis. Curr Biol 15:2038–2043

    Article  CAS  PubMed  Google Scholar 

  15. Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL (2005) Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17:2186–2203

    Article  CAS  PubMed  Google Scholar 

  16. Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ (2005) Elucidation of the small RNA component of the transcriptome. Science 309:1567–1569

    Article  CAS  PubMed  Google Scholar 

  17. 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  Google Scholar 

  18. Lu C, Kulkarni K, Souret FF, MuthuValliappan R, Tej SS, Poethig RS, Henderson IR, Jacobsen SE, Wang W, Green PJ, Meyers BC (2006) MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant. Genome Res 16:1276–1288

    Article  CAS  PubMed  Google Scholar 

  19. Zhu QH, Spriggs A, Matthew L, Fan L, Kennedy G, Gubler F, Helliwell C (2008) A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 18:1456–1465

    Article  CAS  PubMed  Google Scholar 

  20. Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Vazquez F, Voinnet O, Weigel D, Zhu JK (2008) Criteria for annotaion of plant microRNAs. Plant Cell 20:3186–3190

    Article  CAS  PubMed  Google Scholar 

  21. Maroney PA, Chamnongpol S, Souret F, Nilsen TW (2007) A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation. RNA 13:930–936

    Article  CAS  PubMed  Google Scholar 

  22. Moxon S, Moulton V, Kim JT (2008) A scoring matrix approach to detecting miRNA target sites. Algorithms Mol Biol 3:3

    Article  PubMed  Google Scholar 

  23. Zhang Y (2005) miRU: an automated plant miRNA target prediction server. Nucleic Acids Res 33:W701–W704

    Article  CAS  PubMed  Google Scholar 

  24. Souret FF, Kastenmayer JP, Green PJ (2004) AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. Mol Cell 15:173–183

    Article  CAS  PubMed  Google Scholar 

  25. Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, Krizan KA, Carrington JC (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4:205–217

    Article  CAS  PubMed  Google Scholar 

  26. German MA, Pillay M, Jeong DH, Hetawal A, Luo S, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R, De Paoli E, Lu C, Schroth G, Meyers BC, Green PJ (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol 26:941–946

    Article  CAS  PubMed  Google Scholar 

  27. Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309

    Article  CAS  PubMed  Google Scholar 

  28. Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio-Somoza I, Leyva A, Weigel D, Garcia JA, Paz-Ares J (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 39:1033–1037

    Article  CAS  PubMed  Google Scholar 

  29. Ebert MS, Neilson JR, Sharp PA (2007) MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4:721–726

    Article  CAS  PubMed  Google Scholar 

  30. Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264

    Article  CAS  PubMed  Google Scholar 

  31. Claes B, Dekeyser R, Villarroel R, Van den Bulcke M, Bauw G, Van Montagu M, Caplan A (1990) Characterization of a rice gene showing organ-specific expression in response to salt stress and drought. Plant Cell 2:19–27

    Article  CAS  PubMed  Google Scholar 

  32. Moons A, De Keyser A, Van Montagu M (1997) A group 3 LEA cDNA of rice, responsive to abscisic acid, but not to jasmonic acid, shows variety-specific differences in salt stress response. Gene 191:197–204

    Article  CAS  PubMed  Google Scholar 

  33. Wilhelm KS, Thomashow MF (1993) Arabidopsis thaliana cor15b, an apparent homologue of cor15a, is strongly responsive to cold and ABA, but not drought. Plant Mol Biol 23:1073–1077

    Article  CAS  PubMed  Google Scholar 

  34. Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J 16:433–442

    Article  CAS  PubMed  Google Scholar 

  35. Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y, Yoshiwara K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses. Plant Physiol 133:1755–1767

    Article  CAS  PubMed  Google Scholar 

  36. Huang HJ, Fu SF, Tai YH, Chou WC, Huang DD (2002) Expression of Oryza sativa MAP kinase gene is developmentally regulated and stress-responsive. Physiol Plant 114:572–580

    Article  CAS  PubMed  Google Scholar 

  37. Nishizawa A, Yabuta Y, Yoshida E, Maruta T, Yoshimura K, Shigeoka S (2006) Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress. Plant J 48:535–547

    Article  CAS  PubMed  Google Scholar 

  38. Yamanouchi U, Yano M, Lin H, Ashikari M, Yamada K (2002) A rice spotted leaf gene, Spl7, encodes a heat stress transcription factor protein. Proc Natl Acad Sci USA 99:7530–7535

    Article  CAS  PubMed  Google Scholar 

  39. Bariola PA, Howard CJ, Taylor CB, Verburg MT, Jaglan VD, Green PJ (1994) The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J 6:673–685

    Article  CAS  PubMed  Google Scholar 

  40. Yi K, Wu Z, Zhou J, Du L, Guo L, Wu Y, Wu P (2005) OsPTF1, a novel transcription factor involved in tolerance to phosphate starvation in rice. Plant Physiol 138:2087–2096

    Article  CAS  PubMed  Google Scholar 

  41. Hur YJ, Lee HG, Jeon EJ, Lee YY, Nam MH, Yi G, Eun MY, Nam J, Lee JH, Kim DH (2007) A phosphate starvation-induced acid phosphatase from Oryza sativa: phosphate regulation and transgenic expression. Biotechnol Lett 29:829–835

    Article  CAS  PubMed  Google Scholar 

  42. Bari R, Datt Pant B, Stitt M, Scheible WR (2006) PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol 141:988–999

    Article  CAS  PubMed  Google Scholar 

  43. Takahashi H, Yamazaki M, Sasakura N, Watanabe A, Leustek T, Engler JA, Engler G, Van Montagu M, Saito K (1997) Regulation of sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana. Proc Natl Acad Sci USA 94:11102–11107

    Article  CAS  PubMed  Google Scholar 

  44. German MA, Luo S, Schroth G, Meyers BC, Green PJ (2009) Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. Nat Protoc 4:356–362

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The methods in this chapter were developed with support from NSF, USDA, and DOE.

Author information

Authors and Affiliations

  1. Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA

    Dong-Hoon Jeong, Marcelo A. German & Linda A. Rymarquis

  2. Chemistry–Biology Interface Program, Department of Chemistry and Biochemistry, and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA

    Shawn R. Thatcher

  3. Department of Plant and Soil Sciences, School of Marine Science and Policy, and Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA

    Pamela J. Green

Authors
  1. Dong-Hoon Jeong
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  2. Marcelo A. German
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  3. Linda A. Rymarquis
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  4. Shawn R. Thatcher
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  5. Pamela J. Green
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Editor information

Editors and Affiliations

  1. Delaware Biotechnology Institute, University of Delaware, Innovation Way 15, Newark, 19711, U.S.A.

    Blake C. Meyers

  2. Delaware Biotechnology Institute, University of Delaware, Innovation Way 15, Newark, 19711, U.S.A.

    Pamela J. Green

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© 2010 Humana Press, a part of Springer Science+Business Media, LLC

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Jeong, DH., German, M.A., Rymarquis, L.A., Thatcher, S.R., Green, P.J. (2010). Abiotic Stress-Associated miRNAs: Detection and Functional Analysis. In: Meyers, B., Green, P. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 592. Humana Press. https://doi.org/10.1007/978-1-60327-005-2_14

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  • DOI: https://doi.org/10.1007/978-1-60327-005-2_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-004-5

  • Online ISBN: 978-1-60327-005-2

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