Barley primary microRNA expression pattern is affected by soil water availability

  • Aleksandra Swida-Barteczka Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland
  • Katarzyna Kruszka Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland
  • Aleksandra Grabowska Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland
  • Andrzej Pacak Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland
  • Artur Jarmolowski Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland
  • Marzena Kurowska Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
  • Iwona Szarejko Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
  • Zofia Szweykowska-Kulinska Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland
Keywords: pri-miRNA, miRNA, drought, rehydration, barley genotypes


MicroRNAs are short molecules of 21-24 nt length. Present in all eukaryotic organisms, they regulate gene expression by guiding posttranscriptional silencing of mRNAs. In plants, they are key players in signal transduction, growth and development, and response to abiotic and biotic stresses. Barley (Hordeum vulgare) is an economically important monocotyledonous crop plant. Drought is the world’s main cause of loss in cereal production. We have constructed a high-throughput Real-Time RT-qPCR platform for parallel determination of 159 barley primary microRNAs levels. The platform was tested in two drought-and-rehydration-treated barley genotypes (Rolap and Sebastian). We have determined changes in the expression of primary microRNAs responding to mild drought, severe drought, and rehydration. From the results, we conclude that the primary microRNA expression is altered relative to the stress intensity. Mild drought and rehydration mostly decrease the pri-miRNAs levels in both of the tested genotypes. Severe drought mainly induces primary microRNA expression. The main difference between the genotypes tested was a much-stronger induction of pri-miRNAs in Rolap encountering severe drought. The primary microRNAs respond dynamically to mild drought, severe drought, and rehydration treatments. We propose some of the individual pri-miRNAs to be drought stress or rehydration markers. We recommend the primary microRNA RT-qPCR-based platform to be a universal tool for testing the strength of drought response in barley. Hence, the platform can be used to determine drought stress levels applied to barley plants. The usage of the platform in biotechnology is also postulated.


Alaba S, Piszczalka P, Pietrykowska H, Pacak AM, Sierocka I, Nuc PW, Singh K, Plewka P, Sulkowska A, Jarmolowski A, Karlowski WM, Szweykowska-Kulinska Z. (2015) The liverwort Pellia endiviifolia shares microtranscriptomic traits that are common to green algae and land plants. New Phytol 206(1):352-67. doi: 10.1111/nph.13220

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2

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. doi: 10.1105/tpc.016238

Barciszewska-Pacak M, Milanowska K, Knop K, Bielewicz D, Nuc P, Plewka P, Pacak AM, Vazquez F, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2015) Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses. Front Plant Sci 6:410. doi: 10.3389/fpls.2015.00410

Bartel DP (2004) MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116:281-297. doi: 10.1016/S0092-8674(04)00045-5

Bielewicz D, Dolata J, Zielezinski A, Alaba S, Szarzynska B, Szczesniak MW, Jarmolowski J, Szweykowska-Kulinska Z, Karlowski W (2012) mirEX: a platform for comparative exploration of plant pri-miRNA expression data. Nucleic Acid Res 40:191–197. doi: 10.1093/nar/gkr878

Boualem A, Laporte P, Jovanovic M, Laffont C, Plet J, Combier JP, Niebel A, Crespi M, Frugier F (2008) MicroRNA166 controls root and nodule development in Medicago truncatula. The Plant Journal 54:876-887. doi: 10.1111/j.1365-313X.2008.03448.x

Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190. doi: 10.1126/science.1159151

Brown JWS, Marshall DF, Echeverria M (2008) Intronic noncoding RNAs and splicing. Trends Plant Sci 13:335–342. doi: 10.1016/j.tplants.2008.04.010

Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell 136:642-655. doi: 10.1016/j.cell.2009.01.035

Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022-5. doi: 10.1126/science.1088060

Chen X, Zhang Z, Liu D, Zhang K, Li A, Mao L (2010) SQUAMOSA promoter-binding protein-like transcription factors: star players for plant growth and development. J Integr Plant Biol 52:946-51. doi: 10.1111/j.1744-7909.2010.00987.x

Deng W, Nickle DC, Learn GH, Maust B, and Mullins JI (2007) ViroBLAST: A stand-alone BLAST web server for flexible queries of multiple databases and user's datasets. Bioinformatics 23(17):2334-2336. doi: 10.1093/bioinformatics/btm331

Devaux P, Adamski T. Surma M (1992) Inheritance of seed set in crosses of spring barley and Hordeum bulbosum L. Crop Sci 32:269-271. doi: 10.2135/cropsci1992.0011183X003200010054x

Eamens AL, Smith NA, Curtin SJ, Wang MB, Waterhouse PM (2009) The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15:2219–2235. doi: 10.1261/rna.1646909

Hackenberg M, Gustafson P, Langridge P, Shi BJ (2015) Differential expression of microRNAs and other small RNAs in barley between water and drought conditions. Plant Biotechnol J 13, pp. 2–13. doi: 10.1111/pbi.12220

Hofacker IL, Fontana W, Stadler PF, Bonhoeffer LS, Tacker M, Schuster P (1994) Fast folding and comparison of RNA secondary structures (The Vienna RNA Package). Monatsh Chem 125:167–188. doi: 10.1007/BF00818163

International Barley Genome Sequencing Consortium, Mayer KF, Waugh R, Brown JW, Schulman A, Langridge P, Platzer M, Fincher GB, Muehlbauer GJ, Sato K, Close TJ, Wise RP, Stein N (2012) A physical, genetic and functional sequence assembly of the barley genome. Nature Nov 29; 491(7426):711-6. doi: 10.1038/nature11543

Jian X, Zhang L, Li G, Zhang L, Wang X, Cao X, Fang X, Chen F (2010) Identification of novel stress regulated microRNAs from Oryza sativa L. Genomics 95:47–55. doi: 10.1016/j.ygeno.2009.08.017

Kantar M, Unver T, Budak H (2010) Regulation of barley miRNAs upon dehydration stress correlated with target gene expression. Funct Integr Genomics 10:493-507. doi: 10.1007/s10142-010-0181-4

Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298. doi: 10.1093/bib/bbn013

Khraiwesh B, Zhua JK, Zhuc J (2012) Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochim Biophys Acta 1819:137–148. doi: 10.1016/j.bbagrm.2011.05.001

Kozomara A, Griffiths-Jones S (2014) miRBase:annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:D68-73. doi: 10.1093/nar/gkt1181

Kruszka K, Pieczynski M, Windels D, Bielewicz D, Jarmołowski A, Szweykowska-Kulinska Z, Vazques F (2012) Role of microRNAs and Rother sRNAs of plants in their changing environment. J Plant Physiol 169:1664-1672. doi: 10.1016/j.jplph.2012.03.009

Kruszka K, Pacak A, Swida-Barteczka A, Stefaniak AK, Kaja E, Sierocka I, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2013) Developmentally regulated expression and complex processing of barley pri-microRNAs. BMC Genomics 14:34. doi: 10.1186/1471-2164-14-34

Kruszka K, Pacak A, Swida-Barteczka A, Nuc P, Alaba S, Wroblewska Z, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2014) Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley. J Exp Bot 65(20):6123-35. doi: 10.1093/jxb/eru353

Kurihara Y, Takashi Y, Watanabe Y (2006) The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA 12: 206-212. doi: 10.1261/rna.2146906

Lacombe S, Nagasaki H, Santi C, Duval D, Piegu B, Bangratz M, Breitler JC, Guiderdoni E, Brugidou C, Hirsch J, Cao X, Brice C, Panaud O, Karlowski WM, Sato Y, Echeverria M (2008) Identification of precursor transcripts for 6 novel miRNAs expands the diversity on the genomic organisation and expression of miRNA genes in rice. BMC Plant Biol 8:123. doi: 10.1186/1471-2229-8-123

Laubinger S, Sachsenberg T, Zeller G, Busch W, Lohmann JU, Ratsch G, Weigel D (2008) Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and micro RNA processing in Arabidopsis thaliana. Proc Natl Acad Sci USA 105:8795–8800. doi: 10.1073/pnas.0802493105

Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23:4051-4060. doi: 10.1038/sj.emboj.7600385

Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663-70. doi: 10.1093/emboj/cdf476

Li WX, Oono Y, Zhu J, He XJ, Wu JM, Iida K, Lu XY, Cui X, Jin H, Zhu JK (2008) The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance. The Plant Cell 20:2238-2251. doi: 10.1105/tpc.108.059444

Li YP, Ye, W, Wang M, Yan XD (2009) Climate change and drought: a risk assessment of crop-yield impacts. Clim Res 39:31-46. doi: 10.3354/cr00797

Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14:836-843. doi: 10.1261/rna.895308

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. doi: 10.1126/science.1076311

Lv S, Nie X, Wang L, Du X, Biradar SS, Jia X, Weining S (2012) Identification and characterization of MicroRNAs from barley (Hordeum vulgare L.) by high-throughput sequencing. Int J Mol Sci 13:2973–2984. doi: 10.3390/ijms13032973

Mallory AC, Reinhart BJ, Jones-Rhoades MW, Tang G, Zamore PD, Barton MK, Bartel DP (2004) MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5' region. EMBO J 23:3356-64. doi: 10.1038/sj.emboj.7600340

McConnell JR, Emery J, Eshed Y, Bao N, Bowman J, Barton MK (2001) Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411:709-13. doi: 10.1038/35079635

Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by miRNAs. Nature 425:257–263. doi: 10.1038/nature01958

Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102:3691–3696. doi: 10.1073/pnas.0405570102

Pieczynski M, Marczewski W, Hennig J, Dolata J, Bielewicz D, Piontek P, Wyrzykowska A, Krusiewicz D, Strzelczyk-Zyta D, Konopka-Postupolska D, Krzeslowska M, Jarmolowski A, Szweykowska-Kulinska Z (2013) Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato. Plant Biotechnol J 11:459-469. doi: 10.1111/pbi.12032

Raczynska KD, Simpson CG, Ciesiolka A, Szewc L, Lewandowska D, McNicol J, Szweykowska-Kulinska Z, Brown JWS, Jarmolowski A (2010) Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana. Nucleic Acids Res 38:265-278. doi: 10.1093/nar/gkp869

Raczynska KD, Stepien A, Kierzkowski D, Kalak M, Bajczyk M, McNicol J, Simpson CG, Szweykowska-Kulinska Z, Brown JW, Jarmolowski A (2014) The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana. Nucleic Acids Res. 42(2):1224-44. doi: 10.1093/nar/gkt894

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. doi: 10.1101/gad.1476406

Ramakers C, Ruijter JM, Deprez RH, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66. doi: 10.1016/S0304-3940(02)01423-4

Rapacz M, Stepien A, Skorupa K (2012) Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age. Acta Physiol Plant 34:1723–1733. doi: 10.1007/s11738-012-0967-1

Rogers K, Chen X (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25:2383-99. doi: 10.1105/tpc.113.113159

Ru P, Xu L, Ma H, Huang H (2006) Plant fertility defects induced by the enhanced expression of microRNA167. Cell Res 16:457-65. doi: 10.1038/

Schreiber AW, Shi BJ, Huang CY, Langridge P, Baumann U (2011) Discovery of barley miRNAs through deep sequencing of short reads. BMC Genomics 25:129. doi: 10.1186/1471-2164-12-129

Song L, Han MH, Lesicka J, Fedoroff N (2007) Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. Proc Natl Acad Sci USA 104:5437-42. doi: 10.1073/pnas.0701061104

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. doi: 10.1105/tpc.106.041673

Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001-2019. doi: 10.1105/tpc.104.022830

Szarzynska B, Sobkowiak L, Jarmolowski A, Szweykowska-Kulinska Z (2011) Gene structures and processing of plant pri-miRNAs. Res Adv in Nucleic Acids Res 1:1-12

Szarzyńska B, Sobkowiak Ł, Pant BD, Balazadeh S, Scheible WR, Mueller-Roeber B, Jarmołowski A, Szweykowska-Kulińska Z (2009) Gene structure and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs. Nucleic Acids Res 37:3083-3093. doi: 10.1093/nar/gkp189

Szwed M, Karg G, Pińskwar I, Radziejewski M, GraczykD, Kędziora A, Kundzeiwcz ZW (2010) Climate change and its effect on agriculture, water resources and human health sectors in Poland. Nat. Hazards Earth Syst. Sci. 10:1725-1737. doi: 10.5194/nhess-10-1725-2010

Szweykowska-Kulińska Z, Jarmołowski A, Vazquez F (2013) The crosstalk between plant microRNA biogenesis factors and the spliceosome. Plant Signal Behav 8:e26955. doi: 10.4161/psb.26955

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. doi: 10.1111/j.1365-313X.2006.02768.x

Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71-W74. doi: 10.1093/nar/gkm306

Vaucheret H, Vazquez F, Crété 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. doi: 10.1101/gad.1201404

Wu MF, Tian Q, Reed JW (2006) Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development 133:4211-8. doi: 10.1242/dev.02602

Yang L, Liu Z, Lu F, Dong A, Huang H (2006) SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J 47:841-850. doi: 10.1111/j.1365-313X.2006.02835.x

Yang Z, Ebright YW, Yu B, Chen X (2006) HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 20 OH of the 30 terminal nucleotide. Nucleic Acids Res 34:667-675. doi: 10.1093/nar/gkj474

Yu B, Bi L, Zheng B, Ji L, Chevalier D, Agarwal M, Ramachandran V, Li W, Lagrange T, Walker JC, Chen X (2008) The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc Natl Acad Sci USA 105:10073-10078. doi: 10.1073/pnas.0804218105

Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, Steward R, Chen X (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307:932-935. doi: 10.1126/science.1107130

Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Res 14:415-421. doi: 10.1111/j.1365-3180.1974.tb01084.x

Zhang J, Xu Y, Huan Q, Chong K (2009) Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response. BMC Genomics 10:449. doi: 10.1186/1471-2164-10-449

Zhang W, Gao S, Zhou X, Xia J, Chellappan P, Zhou X, Zhang X, Jin H (2010) Multiple distinct small RNAs originate from the same microRNA precursors. Genome Biol 11:R81. doi: 10.1186/gb-2010-11-8-r81

Zhang X, Zou Z, Gong P, Zhang J, ZiafK, Li H, Xiao F, Ye Z (2011) Over-expression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnol Lett 33:403-409. doi: 10.1007/s10529-010-0436-0

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. doi: 10.1186/1471-2199-10-29

Zhao Y, Zhang C, Liu W, Gao W, Liu C, Song G, Li WX, Mao L, Chen B, Xu Y, Li X, Xie C (2016) An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design. Sci Rep. 6:23890. doi: 10.1038/srep23890

Zielezinski A, Dolata J, Alaba S, Kruszka K, Pacak A, Swida-Barteczka A, Knop K, Stepien A, Bielewicz D, Pietrykowska H, Sierocka I, Sobkowiak L, Lakomiak A, Jarmolowski A, Szweykowska-Kulinska Z, Karlowski WM (2015) mirEX 2.0 – an integrated environment for expression profiling of plant microRNAs. BMC Plant Biol 15:144. doi: 10.1186/s12870-015-0533-2