Abstract
Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental–in silico approach was adopted for characterization of two conserved miRNAs, miR166 and miR171, from black pepper (Piper nigrum). A PCR-based detection and cloning strategy of miRNAs from tissues of black pepper was used. Conservation analysis of miR166 and miR171 along with their corresponding targets identified from P. nigrum revealed that these miRNAs are highly conserved with their counterparts in other plant species. miRNA-mediated cleavage of the conserved targets was also verified by RLM-RACE experiments. Real-time quantitative PCR revealed the differential expression patterns of these miRNAs in black pepper tissues. Our miRNA-based phylogenetic analysis of plants belonging to the Piperaceae family was in agreement with the typical paleoherb evolutionary scheme of primitive angiosperms. This method will help in the detection of evolutionarily conserved miRNAs in other plant species and provide a strategy for a novel phylogenetic reconstruction based on the evolutionary history of miRNA genes.
Similar content being viewed by others
References
Adai A, Johnson C, Mlotshwa S, Archer-Evans S, Manocha V, Vance V, Sundaresan V (2005) Computational prediction of miRNAs in Arabidopsis thaliana. Genome Res 15:78–91
Allen E, Xie ZX, Gustafson AM, Sung GH, Spatafora JW, Carrington JC (2004) Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat Genet 36:1282–1290
Axtell MJ, Bartel DP (2005) Antiquity of microRNAs and their targets in land plants. Plant Cell 17:1658–1673
Axtell MJ, Westholm JO, Lai EC (2011) Vive la différence: biogenesis and evolution of microRNAs in plants and animals. Genome Biol 12:221
Bartel D (2004) MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116:281–297
Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA 102:11928–11933
Bolle C (2004) The role of GRAS proteins in plant signal transduction and development. Planta 218:683–692
Bonnet E, Wuyts J, Rouze P, Van de Peer Y (2004) Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identify important target genes. Proc Natl Acad Sci USA 101:11511–11516
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. Plant J 54:876–887
Byrne ME (2006) Shoot Meristem Function and Leaf Polarity: The role of class III HD–ZIP genes. PLoS Genet 2(6):e89
Carlsbecker A, Lee JY, Roberts CJ et al (2010) Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465:316–321
Chang TH, Horng JT, Huang HD (2008) RNALogo: a new approach to display structural RNA alignment. Nucleic Acids Res 36:W91–W96
Chen X (2005) microRNA biogenesis and function in plants. FEBS Lett 579:5923–5931
Chen C, Ridzon D, Broomer AJ, Zhou Z, 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 33:179
Christopher M, Lincoln S, Doreen W (2006) Evolution of Arabidopsis microRNA families through duplication events. Genome Res 16:510–519
Cuperus JT, Carbonell A, Fahlgren N, Garcia-Ruiz H, Burke RT, Takeda A, Sullivan CM, Gilbert SD, Montgomery TA, Carrington JC (2010) Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nat Struct Mol Biol 17:997–1003
Cuperus JT, Fahlgren N, Carrington JC (2011) Evolution and functional diversification of MIRNA genes. Plant Cell 23:431–442
Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39:w155–w159
De Felippes FF, Schneeberger K, Dezulian T, Huson DH, Weigel D (2008) Evolution of Arabidopsis thaliana microRNAs from random sequences. RNA 14:2455–2459
Dezulian T, Remmert M, Palatnik JF, Weigel D, Huson DH (2006) Identification of plant microRNA homologs. Bioinformatics 22:359–360
Donaire L, Pedrola L, de la Rosa R, Llave C (2011) High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L.). PLoS One 6:e27916
Emery JF, Floyd SK, Alvarez J, Eshed Y, Hawker NP, Izhaki A (2003) Radial patterning of Arabidopsis shoots by Class III HD-ZIP and KANADI genes. Curr Biol 13:1768–1774
Fattash I, Reski BR, Hess WR, Frank W (2007) Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution. BMC Plant Biol 7:13
Floyd SK, Bowman JL (2004) Ancient microRNA regulation of gene expression in land plants. Nature 428:485–486
Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140–D144
Hawker NP, Bowman JL (2004) Underground Polarity: Roles for Class III HD-Zip and KANADI genes in Arabidopsis root development. Plant Physiol 135:2261–2270
Jagadeeswaran G, Nimmakayala P, Zheng Y, Gowdu K, Reddy UK, Sunkar R (2012) Characterization of the small RNA component of leaves and fruits from four different cucurbit species. BMC Genom 13:329
Jaramillo MA, Manos PS (2001) Phylogeny and patterns of floral diversity in the genus Piper (Piperaceae). Am J Bot 88:706–716
Jay F, Renou JP, Voinnet O, Navarro L (2010) Biotic stress-associated microRNAs: identification, detection, regulation, and functional analysis. Methods Mol Biol 592:183–202
Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress induced miRNA. Mol Cell 14:787–799
Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53
Joy N, Soniya EV (2012) Identification of a miRNA candidate reflects the possible significance of transcribed microsatellites in the hairpin precursors of black pepper. Funct Integr Genomics 12:387–395
Juarez MT, Kui JS, Thomas J, Heller BA, Timmermans MC (2004) microRNA-mediated repression of rolled leaf specifies maize leaf polarity. Nature 428:84–88
Jung JH, Park CM (2007) MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis. Planta 225:1327–1338
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
Kidner CA, Martienssen RA (2004) Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428:81–84
Kim J, Jung JH, Reyes JL, Kim YS, Kim SY, Chung KS, Kim JA, Lee M, Lee Y, Kim VN, Chua NH, Park CM (2005) MicroRNA-directed cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems. Plant J 42:84–94
Krüger J, Rehmsmeier M (2006) RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res 34:W451–W454
Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S, Kim V (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419
Lee MH, Kim B, Song SK, Heo JO, Yu NI, Lee SA, Kim M, Kim DG, Sohn SO, Lim CE, Chang KS, Lee MM, Lim J (2008) Large-scale analysis of the GRAS gene family in Arabidopsis thaliana. Plant Mol Biol 67:659–670
Li A, Mao L (2007) Evolution of plant microRNA gene families. Cell Res 17:212–218
Li H, Xu L, Wang H, Yuan Z, Cao X, Yang Z, Zhang D, Xu Y, Huang H (2005) The putative RNA-dependent RNA polymerase RDR6 acts synergistically with ASYMMETRIC LEAVES1 and 2 to repress BREVIPEDICELLUS and microRNA165/166 in Arabidopsis leaf development. Plant Cell 17:2157–2171
Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619
Llave C, Franco-Zorrilla JM, Solano R, Barajas D (2011) Target validation of plant microRNAs. Methods Mol Biol 732:187–208
Mallory AC, Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet 38:850
Manohar S, Jagadeeswaran G, Nimmakayala P, Tomason Y, Almeida A, Sunkar R, Levi A, Reddy UK (2012) Dynamic regulation of novel and conserved miRNAs across various tissues of diverse Cucurbit species. Plant Mol Biol Rep. doi:10.1007/s11105-012-0506-7
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 411:709–713
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, Voinnet O, Watanabe Y, Weigel D, Zhu JK (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3790
Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C et al (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 50 terminal nucleotide. Cell 133:116–127
Molnár A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447:1126–1129
Nozawa M, Miura S, Nei M (2012) Origins and evolution of microRNA genes in plant species. Genome Biol Evol 4(3):230–23
Pang M, Woodward AW, Agarwal V, Guan X, Ha M, Ramachandran V, Chen X, Triplett BA, Stelly DM, Chen ZJ (2009) Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.). Genome Biol 10:R122
Parizotto EA, Dunoye P, Rahm N, Himber C, Vionnet O (2004) In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18:2237–2242
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
Piriyapongsa J, Jordan IK (2008) Dual coding of siRNAs and miRNAs by plant transposable elements. RNA 14:814–821
Pysh LD, Wysocka-Diller JW, Camilleri C, Bouchez D, Benfey PN (1999) The GRAS gene family in Arabidopsis: Sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J 18:111–119
Rehmsmeier M, Steffen P, Höchsmann M, Giegerich R (2004) Fast and effective prediction of microRNA/target duplexes RNA 10:1507–1517
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626
Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520
Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527
Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Method 3:12
Wang XJ, Reyes JL, Chua NH, Gaasterland T (2004) Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets. Genome Biol 5:1–15
Wang L, Mai YX, Zhang YC, Luo Q, Yang HQ (2010) MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis. Mol Plant 3:794–806
Williams L, Grigg SP, Xie M, Christensen S, Fletcher JC (2005) Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes. Development 132:3657–3668
Wong CE, Zhao YT, Wang XJ, Croft L, Wang ZH, Haerizadeh F, Mattick JS, Singh MB, Carroll BJ, Bhalla PL (2011) MicroRNAs in the shoot apical meristem of soybean. J Exp Bot 62:2495–2506
Yang Y, Chen X, Chen J, Xu H, Li J, Zhang Z (2011) Identification of novel and conserved microRNAs in Rehmannia glutinosa L. by Solexa sequencing. Plant Mol Biol Rep 29:986–996
Yao Y, Guo G, Ni Z, Sunkar R, Du J, Zhu JK, Sun Q (2007) Cloning and characterisation of microRNAs from wheat (Triticum aestivum L.). Genome Biol 8:R96
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
Zhang B, Pan X, Cannon CH, Cobb GP, Anderson TA (2006a) a) Conservation and divergence of plant microRNA genes. Plant J 46:243–259
Zhang BH, Pan XP, Anderson TA (2006b) Identification of 188 conserved maize microRNAs and their targets. FEBS Lett 580:3753–3762
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
Zhang Y, Jiang W, Gao L (2011) Evolution of microRNA genes in Oryza sativa and Arabidopsis thaliana: an update of the inverted duplication model. PLoS One 6:e28073
Zhao T, Li G, Mi S, Li S, Hannon GJ, Wang XJ, Qi Y (2007) A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii. Genes Dev 21:1190–1203
Zhou GK, Kubo M, Zhong R, Demura T, Ye ZH (2007) Overexpression of miR165 affects apical meristem formation, organ polarity establishment and vascular development in Arabidopsis. Plant Cell Physiol 48:391–404
Zhu H, Hu F, Wang R, Zhou X, Sze SH, Liou LW, Barefoot A, Dickman M, Zhang X (2011) Arabidopsis argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145:242–256
Zuker M, Mathews DH, Turner DH (1999) Algorithms and thermodynamics for RNA secondary structure prediction:a practical guide. In: Barciszewski J, Clark BFC (ed) RNA biochemistry and biotechnology, NATO ASI series, Kluwer, Dordrecht
Acknowledgments
The authors greatly acknowledge the award of a junior research fellowship from Council of Scientific and Industrial Research (CSIR), New Delhi, India, and financial support from Department of Biotechnology, Government of India.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Asha, S., Nisha, J. & Soniya, E.V. In silico Characterisation and Phylogenetic Analysis of Two Evolutionarily Conserved miRNAs (miR166 and miR171) from Black Pepper (Piper nigrum L.). Plant Mol Biol Rep 31, 707–718 (2013). https://doi.org/10.1007/s11105-012-0532-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11105-012-0532-5