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Identification and validation of conserved microRNAs along with their differential expression in roots of Vigna unguiculata grown under salt stress

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Abstract

MicroRNAs (miRNAs) are 20–24 nucleotide long non-coding RNAs known to play important regulatory roles during plant development, organ morphogenesis, and stress responses by controlling gene expression. Although Vigna unguiculata (cowpea) is an economically important salt sensitive member of legumes, very little is known about the conserved miRNAs and their expression profile during salinity stress in this plant. In the present study using comparative genomic approach and following a set of strict filtering criteria we have identified 18 conserved V. unguiculata miRNAs belonging to 16 distinct miRNA families. Using these potential miRNA sequences 15 potential target genes were predicted and all of them were identified as transcription factors. Seven of these predicted V. unguiculata miRNAs were experimentally validated in the root tissues and found to be up-regulated during salt stress as revealed by quantitative real time PCR (qRT-PCR). Perfectly cleaved Auxin response factor (ARF), the target transcript of V. unguiculata miR160 was detected successfully by modified 5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method.

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Abbreviations

EST:

Expressed sequence tag

GSS:

Genomic survey sequence

RISC:

RNA induced silencing complex

MFE:

Minimum folding free energy

MFEI:

Minimum folding free energy index

RLM-RACE:

RNA ligase-mediated rapid amplification of cDNA ends

qRT-PCR:

Quantitative real time PCR

DFCI:

Dana Farber Cancer Institute

SBP:

Squamosa promoter binding protein

SPL:

Squamosa promoter binding protein-like protein

ARF:

Auxin response factor

TCP:

Teosinte branched1-Cycloidea-Pcf

CBF:

CCAAT-binding transcription factor

NFY:

Nuclear factor Y

AP2:

APETALA2

AGO1:

Argonaute1

TC:

Tentative contigs

snRNA:

Small nuclear RNA

References

  • Altschul S, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Arenas-Huertero C, Pérez B, Rabanal F, Blanco-Melo D, De la Rosa C, Estrada Navarrete G, Sanchez F, Covarrubias AA, Reyes JL (2009) Conserved and novel miRNAs in the legume Phaseolus vulgaris in response to stress. Plant Mol Biol 70:385–401

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Bayuelo-Jimenez JS, Craig R, Lynch JP (2002) Salinity tolerance of Phaseolus species during germination and early seedling growth. Crop Sci 42:1584–1594

    Article  Google Scholar 

  • Bonnet E, Wuyts J, Rouze′ P, Van de Peer Y (2004) Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. Proc Natl Acad Sci USA 101:11511–11516

    Article  PubMed  CAS  Google Scholar 

  • Bressani R (1985) Nutritive value of cowpea. In: Singh SR, Rachie KO (eds) Cowpea research, production and utilization. Wiley, New York, p 359

    Google Scholar 

  • Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Chen C, Tao C, Peng H, Ding Y (2007) Genetic analysis of salt stress responses in Asparagus Bean (Vigna unguiculata (L.) ssp. sesquipedalis Verdc.). J Hered 98:655–665

    Article  PubMed  CAS  Google Scholar 

  • Chen R, Hu Z, Zhang H (2009) Identification of MicroRNAs in Wild Soybean (Glycine soja). J Integr Plant Biol 51:1071–1079

    Article  PubMed  CAS  Google Scholar 

  • Ding D, Zhang L, Wang H, Liu Z, Zhang Z, Zheng Y (2009) Differential expression of miRNAs in response to salt stress in maize roots. Ann Bot 103:29–38

    Article  PubMed  CAS  Google Scholar 

  • Dong J, Kim ST, Lord EM (2005) Plantacyanin plays a role in reproduction in Arabidopsis. Plant Physiol 138:778–789

    Article  PubMed  CAS  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  Google Scholar 

  • Feng J, Wang K, Liu X, Chen S, Chen J (2009) The quantification of tomato microRNAs response to viral infection by stem-loop real-time RT-PCR. Gene 437:14–21

    Article  PubMed  CAS  Google Scholar 

  • Gusmaroli G, Tonelli C, Mantovani R (2001) Regulation of the CCAAT-Binding NF-Y subunits in Arabidopsis thaliana. Gene 264:173–185

    Article  PubMed  CAS  Google Scholar 

  • Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Circular 347. The College of Agriculture, University of California, Berkeley

    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  PubMed  CAS  Google Scholar 

  • Kidner CA, Martienssen RA (2005) The developmental role of microRNA in plants. Curr Opin Plant Biol 8:38–44

    Article  PubMed  CAS  Google Scholar 

  • Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101:12753–12758

    Article  PubMed  CAS  Google Scholar 

  • Li Y, Li W, Jin YX (2005) Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa. Acta Biochim Biophys Sin (Shanghai) 37:75–87

    Article  Google Scholar 

  • Liang XF, 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  Google Scholar 

  • Lin SL, Chang D, Ying SY (2005) Asymmetry of intronic pre-miRNA structures in functional RISC assembly. Gene 356:32–38

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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  PubMed  CAS  Google Scholar 

  • Lu S, Sun YH, Chiang VL (2008) Stress-responsive microRNAs in Populus. Plant J 55:131–151

    Article  PubMed  CAS  Google Scholar 

  • Mallory AC, Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet 38:S31–S36

    Article  PubMed  CAS  Google Scholar 

  • Mallory AC, Bartel DP, Bartel B (2005) MicroRNA-directed regulation of Arabidopsis Auxin Response Factor17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17:1360–1375

    Article  PubMed  CAS  Google Scholar 

  • Martins LMV, Xavier GR, Rangel FW, Ribeiro JRA, Neves MCP, Morgado LB, Rumjanek NG (2003) Contribution of biological nitrogen fixation to cowpea: a strategy for improving grain yield in the semi-arid region of Brazil. Biol Fertil Soils 38:333–339

    Article  Google Scholar 

  • Meyers BC, Axtell MJ, Bartel B et al (2008) Criteria for annotation of plant MicroRNAs. Plant Cell 20:3186–3190

    Article  PubMed  CAS  Google Scholar 

  • Munns R (1993) Physiological processes limiting plant growth in saline soils: some dogmas and hypothesis. Plant Cell Environ 16:15–24

    Article  CAS  Google Scholar 

  • Pule-Meulenberg F, Belane AK, Krasova-Wade T, Dakora FD (2010) Symbiotic functioning and bradyrhizobial biodiversity of cowpea (Vigna unguiculata L.Walp.) in Africa. BMC Microbiol. doi:10.1186/1471-2180-10-89

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

    Article  PubMed  CAS  Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108

    Article  PubMed  CAS  Google Scholar 

  • Schwarz S, Grande AV, Bujdoso N, Saedler H, Huijser P (2008) The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis. Plant Mol Biol 67:183–195

    Article  PubMed  CAS  Google Scholar 

  • Singh BB, MohanRaj DR, Dashiell KE, Jackai LEN (1997) Advances in cowpea research. IITA-JIRCAS, Ibadan

    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  PubMed  CAS  Google Scholar 

  • 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  PubMed  CAS  Google Scholar 

  • Wang W, Vinocur B, Altman A (2003) Plant responses to drought, salinity and extreme temperature towards genetic engineering for stress tolerance. Planta 218:1–14

    Article  PubMed  CAS  Google Scholar 

  • Wang XJ, Reyes JL, Chua NH, Gaasterland T (2004) Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets. Genome Biol 5:R65

    Article  PubMed  Google Scholar 

  • Wang Y, Li P, Cao X, Wang X, Zhang A, Li X (2009) Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules. Biochem Biophys Res Commun 378:799–803

    Article  PubMed  CAS  Google Scholar 

  • Wei B, Cai T, Zhang R, Li A, Huo N, Li S, Gu QY, Vogel J, Jia J, Qi Y, Mao L (2009) Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv. Funct Integr Genomics 9:499–511

    Article  PubMed  CAS  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  PubMed  CAS  Google Scholar 

  • Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19:2164–2175

    Article  PubMed  CAS  Google Scholar 

  • Zeng C, Wang W, Zheng Y, Chen X, Bo W, Song S, Zhang W, Peng M (2009) Conservation and divergence of microRNAs and their functions in Euphorbiaceous plants. Nucleic Acids Res 38:981–995

    Article  PubMed  Google Scholar 

  • Zhang BH, Pan X, Cobb GP, Anderson TA (2006a) Plant microRNA: a small regulatory molecule with big impact. Dev Biol 289:3–6

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Zhang B, Wang QL, Pan XP (2007) MicroRNAs and their regulatory roles in animals and plants. J Cell Physiol 210:279–289

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Wei L, Zou X, Tao Y, Liu Z, Zheng Y (2008b) Submergence-responsive MicroRNAs are potentially involved in the regulation of morphological and metabolic adaptations in maize root cells. Ann Bot 102:509–519

    Article  PubMed  CAS  Google Scholar 

  • 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

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are thankful to the Director, Bose Institute for providing the lab facilities; we also thank the Department of Biotechnology, India, for the financial assistance (Sanction no. BT/01/COE/06/03), a SRF to AK and a RA to SP.

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  1. Division of Plant Biology, Bose Institute, Kolkata, 700054, India

    Sujay Paul, Anirban Kundu & Amita Pal

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  1. Sujay Paul
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  2. Anirban Kundu
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  3. Amita Pal
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Correspondence to Amita Pal.

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Paul, S., Kundu, A. & Pal, A. Identification and validation of conserved microRNAs along with their differential expression in roots of Vigna unguiculata grown under salt stress. Plant Cell Tiss Organ Cult 105, 233–242 (2011). https://doi.org/10.1007/s11240-010-9857-7

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