Abstract
Key message
Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.).
Abstract
Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.
Similar content being viewed by others
References
Allen E, Xie Z, 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
Asha S, Nisha J, Soniya EV (2013) In silico characterisation and phylogenetic analysis of two evolutionarily conserved miRNAs (miR166 and miR171) from black pepper (Piper nigrum L.). Plant Mol Biol Rep 31:707–771
Chang TH, Horng JT, Huang HD (2008) RNALogo: a new approach to display structural RNA alignment. Nucleic Acids Res 36:W91–W96
Chao YT, Su CL, Jean WH, Chen WC, Chang YCA, Shih MC (2013) Identification and characterization of the microRNA transcriptome of a moth orchid Phalaenopsis aphrodite. Plant Mol Biol 84:529–548
Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39:9–155
de Felippes FF, Schneeberger K, Dezulian T, Huson DH, Weigel D (2008) Evolution of Arabidopsis thaliana microRNAs from random sequences. RNA 14:2455–2459
Fahlgren N, Jogdeo S, Kasschau KD, Sullivan CM, Chapman EJ, Laubinger S, Smith LM, Dasenko M, Givan SA, Weigel D, Carrington JC (2010) MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana. Plant Cell 22:1074–1089
Galli V, Guzman F, de Oliveira LFV, Loss-Morais G, Korbes AP, Silva SDA, Margis-Pinheiro MAN, Margis R (2014) Identifying MicroRNAs and transcript targets in Jatropha seeds. PLoS One 9:e83727
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
Joy N, Asha S, Mallika V, Soniya EV (2013) De novo transcriptome sequencing reveals a considerable bias in the incidence of simple sequence repeats towards the downstream of ‘Pre-miRNAs’ of black pepper. PLoS One 8:e56694
Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 39:D152–D157
Li R, Yu C, Li Y, Lam TM, Yiu SM, Kristiansen K, Wang J (2009) SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 25:1966–1967
Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619
Lu C, Meyers BC, Green PJ (2007) Green construction of small RNA cDNA libraries for deep sequencing. Methods 43:110–117
Mandhan V, Kaur J, Singh K (2012) smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni. BMC Plant Biol 12:197
Meghwal M, Goswami TK (2012) Nutritional constituent of black pepper as medicinal molecules: a review. Sci Rep 1:129
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
Neilsen CT, Goodall GT, Bracken CP (2012) IsomiRs: the overlooked repertoire in the dynamic microRNAome. Trends Genet 28:544–549
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
Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig PS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102:3691–3696
Piriyapongsa J, Jordan IK (2008) Dual coding of siRNAs and miRNAs by plant transposable elements. RNA 14:814–821
Shen J, Xie K, Xiong L (2010) Global expression profiling of rice microRNAs by one-tube stem-loop reverse transcription quantitative PCR revealed important roles of microRNAs in abiotic stress responses. Mol Genet Genomics 284:477–488
Shivaprasad PV, Chen HM, Patel K, Bond DM, Santos BA, Baulcombe DC (2012) A microRNA superfamily regulates nucleotide binding site—leucine-rich repeats and other mRNAs. Plant Cell 24:859–874
Sunkar R, Li YF, Jagadeeswaran G (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci 17:196–203
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
Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687
Wan LC, Zhang H, Lu S, Zhang L, Qiu Z, Zhao Y, Zeng QY, Lin J (2012) Transcriptome-wide identification and characterization of miRNAs from Pinus densata. BMC Genomics 13:132
Wang L, Liu H, Li D, Chen H (2011) Identification and characterization of maize microRNAs involved in the very early stage of seed germination. BMC Genomics 12:154
Wu B, Wang M, Ma Y, Yuan L, Lu S (2012) High-throughput sequencing and characterization of the small RNA transcriptome reveal features of novel and conserved microRNAs in Panax ginseng. PLoS One 7:e44385
Xin M, Wang Y, Yao Y, Xie C, Peng H, Ni Z, Sun Q (2010) Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biol 10:123–134
Xu W, Cui Q, Li F, Liu A (2013) Transcriptome-wide identification and characterization of microRNAs from Castor Bean (Ricinus communis L.). PLoS One 8:e69995
Zhang BH, Pan XP, Cox SB, Cobb GP, Anderson TA (2006) Evidence that miRNAs are different from other RNAs Cell Mol Life. Sci 63:246–254
Zhang R, Marshall D, Bryan GJ, Hornyik C (2013) Identification and characterization of miRNA transcriptome in potato by high-throughput sequencing. PLoS One 8:e57233
Zhao JP, Jiang XL, Zhang BY, Su XH (2012) Involvement of microRNA-mediated gene expression regulation in the pathological development of stem canker disease in Populus trichocarpa. PLoS One 7(9):e44968
Zuker M, Mathews DH, Turner DH (1999) Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide in RNA biochemistry and biotechnology. In: Barciszewski J, Clark BFC (eds) NATO ASI series. Kluwer Academic Publishers, Dordrecht, The Netherlands, p 370
Acknowledgments
S. Asha gratefully acknowledges the Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR) New Delhi, Government of India. This work was financially supported by Department of Biotechnology, Government of India, New Delhi.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Statement of human rights
This article does not contain any studies with human participants performed by any of the authors.
Informed consent
This article does not contain any studies with animals performed by any of the authors.
Statement on the welfare of animals
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Z. Zhang.
Electronic supplementary material
Below is the link to the electronic supplementary material.
299_2015_1866_MOESM2_ESM.pdf
Supplementary material 2 Fig. S2 Precursor structures of conserved miRNAs predicted from black pepper transcriptome Hair-pin secondary structure is highlighted with mature miRNA (red colour) and star sequence (blue colour). The MFE values of the predicted precursors were also mentioned. (PDF 199 kb)
299_2015_1866_MOESM3_ESM.pdf
Supplementary material 3 Fig. S3 miRNA isoform members of known miRNA families, their reads and alignments to corresponding precursors MiRNA candidates located in the 5p and 3p are numbered. Mature miRNA and miRNA* are highlighted with red and blue colours (PDF 302 kb)
Rights and permissions
About this article
Cite this article
Asha, S., Sreekumar, S. & Soniya, E.V. Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling. Plant Cell Rep 35, 53–63 (2016). https://doi.org/10.1007/s00299-015-1866-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-015-1866-x