Plant Cell Reports

, Volume 36, Issue 11, pp 1815–1827 | Cite as

Identification and expression analysis of microRNAs during ovule development in rice (Oryza sativa) by deep sequencing

  • Ya Wu
  • Liyu Yang
  • Meiling Yu
  • Jianbo Wang
Original Article


Key message

MicroRNA (miRNA) expression profiles during rice ovule development revealed the possible miRNA-mediated regulation between ovule sporophytic tissue and female gametophyte and the involvement of miRNAs in programmed cell death.


MiRNAs are 20–24-nucleotide small RNAs that play key roles in the regulation of many growth and developmental processes in plants. Rice ovule development comprises a series of biological events, which are regulated by complex molecular mechanisms. To gain insight into miRNA-mediated regulation of rice ovule development, Illumina sequencing was used to examine the expression of miRNAs from the megaspore mother cell meiosis stage to the fertilized ovule stage. Based on the sequencing data, 486 known and 204 novel miRNAs were identified during rice ovule development. Moreover, 56, 65 and 11 differentially expressed miRNAs between adjacent developmental stages were identified. By analyzing transcriptome and degradome data, we identified 41, 65 and 12 coherent target genes for the differentially expressed miRNAs in ovule development. We found that changes in the expression of plant hormone-related miRNAs may play important roles in embryo sac development, providing evidence for cross-talk communication between sporophytic tissue and the female gametophyte. Additionally, we revealed that miRNAs may be involved in programmed cell death after fertilization. Finally, we constructed miRNA-mediated regulatory networks that are active during rice ovule development.


Rice ovule development High-throughput sequencing MiRNA expression Integrating expression analysis Programmed cell death 



Gibberellic acid


Growth responding factor


Minimum free energy


Programmed cell death


Transcripts per million


Web gene ontology annotation plot



This work was supported by the State Key Basic Research and Development Plan of China (2013CB126900).

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflicts of interest with this work.

Supplementary material

299_2017_2196_MOESM1_ESM.pdf (1.5 mb)
Supplementary material 1 (PDF 1509 kb)
299_2017_2196_MOESM2_ESM.xls (24 kb)
Supplementary material 2 (XLS 24 kb)
299_2017_2196_MOESM3_ESM.xls (19 kb)
Supplementary material 3 (XLS 19 kb)
299_2017_2196_MOESM4_ESM.xls (886 kb)
Supplementary material 4 (XLS 886 kb)
299_2017_2196_MOESM5_ESM.xls (40 kb)
Supplementary material 5 (XLS 40 kb)
299_2017_2196_MOESM6_ESM.xls (90 kb)
Supplementary material 6 (XLS 90 kb)
299_2017_2196_MOESM7_ESM.xls (17 kb)
Supplementary material 7 (XLS 17 kb)


  1. Allen E, Xie Z, Gustafson AM, Carrington JC (2015) MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121:207–221CrossRefGoogle Scholar
  2. Alonso-Peral MM, Li J, Li Y, Allen RS, Schnippenkoetter W, Ohms S, White RG, Millar AA (2010) The microRNA159-regulated GAMYB-like genes inhibit growth and promote programmed cell death in Arabidopsis. Plant Physiol 154:757–771CrossRefPubMedPubMedCentralGoogle Scholar
  3. Asha S, Sreekumar S, Soniya EV (2016) Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling. Plant Cell Rep 35:53–63CrossRefPubMedGoogle Scholar
  4. Aya K, Ueguchi-Tanaka M, Kondo M, Hamada K, Yano K, Nishimura M, Matsuoka M (2009) Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. Plant Cell 21:1453–1472CrossRefPubMedPubMedCentralGoogle Scholar
  5. Baucher M, Moussawi J, Vandeputte OM, Monteyne D, Mol A, Pérez-Morga D, EI Jaziri M (2013) A role for the miR396/GRF network in specification of organ type during flower development, as supported by ectopic expression of Populus trichocarpa miR396c in transgenic tobacco. Plant Biol 15:892–898CrossRefPubMedGoogle Scholar
  6. Bencivenga S, Colombo L, Masiero S (2011) Cross talk between the sporophyte and the megagametophyte during ovule development. Sex Plant Reprod 24:113–121CrossRefPubMedGoogle Scholar
  7. Chen L, Luan Y, Zhai J (2015) Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Rep 34:2013–2025CrossRefPubMedGoogle Scholar
  8. Cheng CY, Mathews DE, Schaller GE, Kieber JJ (2013) Cytokinin-dependent specification of the functional megaspore in the Arabidopsis female gametophyte. Plant J 73:929–940CrossRefPubMedGoogle Scholar
  9. Curaba J, Singh MB, Bhalla PL (2014) miRNAs in the crosstalk between phytohormone signalling pathways. J Exp Bot 65:1425–1438CrossRefPubMedGoogle Scholar
  10. De Jong M, Wolters-Arts M, Feron R, Mariani C, Vriezen WH (2009) The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development. Plant J 57:160–170CrossRefPubMedGoogle Scholar
  11. Gardner PP, Daub J, Tate JG, Nawrocki EP, Kolbe DL, Lindgreen S, Wilkinson AC, Finn RD, Griffiths-Jones S, Eddy SR, Bateman A (2009) Rfam: updates to the RNA families database. Nucl Acids Res 37:D136–D140CrossRefPubMedGoogle Scholar
  12. Goetz M, Vivian-Smith A, Johnson SD, Koltunow AM (2006) AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis. Plant Cell 18:1873–1886CrossRefPubMedPubMedCentralGoogle Scholar
  13. Guilfoyle TJ, Hagen G (2007) Auxin response factors. Curr Opin Plant Biol 10:453–460CrossRefPubMedGoogle Scholar
  14. Guo HS, Xie Q, Fei JF, Chua NH (2015) MicroRNA directs mRNA cleavage of the transcription factor NAC1 to down-regulate auxin signals for Arabidopsis lateral root development. Plant Cell 17:1376–1386CrossRefGoogle Scholar
  15. Gutierrez L, Bussell JD, Pacurar DI, Schwambach J, Pacurar M, Bellini C (2009) Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance. Plant Cell 21:3119–3132CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hewezi T, Baum TJ (2012) Complex feedback regulations govern the expression of miRNA396 and its GRF target genes. Plant Signal Behav 7:749–751CrossRefPubMedPubMedCentralGoogle Scholar
  17. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53CrossRefPubMedGoogle Scholar
  18. Kim JH, Tsukaya H (2015) Regulation of plant growth and development by the GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR duo. J Exp Bot 66:6093–6107CrossRefGoogle Scholar
  19. Kim JH, Woo HR, Kim J, Lim PO, Lee IC, Choi SH, Hwang D, Nam HG (2009) Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323:1053–1057CrossRefPubMedGoogle Scholar
  20. Kim B, Yu HJ, Park SG, Shin JY, Oh M, Kim N, Mun JH (2012) Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing. BMC Plant Biol 12:21CrossRefGoogle Scholar
  21. Kubo T, Fujita M, Takahashi H, Nakazono M, Tsutsumi N, Kurata N (2013) Transcriptome analysis of developing ovules in rice isolated by laser microdissection. Plant Cell Physiol 54:750–765CrossRefPubMedGoogle Scholar
  22. Kumar R, Tyagi AK, Sharma AK (2011) Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development. Mol Genet Genom 285:245–260CrossRefGoogle Scholar
  23. Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24:713–714CrossRefPubMedGoogle Scholar
  24. Li ZF, Zhang YC, Chen YQ (2015) miRNAs and lncRNAs in reproductive development. Plant Sci 238:46–52CrossRefPubMedGoogle Scholar
  25. Li X, Shahid MQ, Xia J, Lu Z, Fang N, Wang L, Wu J, Chen Z, Liu X (2017) Analysis of small RNAs revealed differential expressions during pollen and embryo sac development in autotetraploid rice. BMC Genom 18:129CrossRefGoogle Scholar
  26. Liu D, Song Y, Chen Z, Yu D (2009) Ectopic expression of miR396 suppresses GRF target gene expression and alters leaf growth in Arabidopsis. Physiol Plantarum 136:223–236CrossRefGoogle Scholar
  27. Liu S, Li JH, Wu J, Zhou KR, Zhou H, Yang JH, Qu LH (2015) StarScan: a web server for scanning small RNA targets from degradome sequencing data. Nucl Acids Res 43:W480–W486CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lopez-Dee ZP, Wittich P, Pe ME, Rigola D, Del Buono I, Gorla MS, Kater MM, Colombo L (1999) OsMADS13, a novel rice MADS-box gene expressed during ovule development. Dev Genet 25:237–244CrossRefPubMedGoogle Scholar
  29. Ma X, Shao C, Wang H, Jin Y, Meng Y (2013) Construction of small RNA-mediated gene regulatory networks in the roots of rice (Oryza sativa). BMC Genom 14:510CrossRefGoogle Scholar
  30. Mecchia MA, Debernardi JM, Rodriguez RE, Schommer C, Palatnik JF (2013) MicroRNA miR396 and RDR6 synergistically regulate leaf development. Mech Dev 130:2–13CrossRefPubMedGoogle Scholar
  31. Meng Y, Shao C, Wang H, Ma X, Chen M (2013) Construction of gene regulatory networks mediated by vegetative and reproductive stage-specific small RNAs in rice (Oryza sativa). N Phytol 197:441–453CrossRefGoogle Scholar
  32. Miyoshi K, Ito Y, Serizawa A, Kurata N (2003) OsHAP3 genes regulate chloroplast biogenesis in rice. Plant J 36:532–540CrossRefPubMedGoogle Scholar
  33. Nagpal P, Ellis CM, Weber H, Ploense S, Barkawi LS, Guilfoyle TJ, Hagen G, Alonso JM, Cohen JD, Farmer EE, Ecker JR, Reed JW (2015) Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation. Development 132:4107–4118CrossRefGoogle Scholar
  34. Pagnussat GC, Alandete-Saez M, Bowman JL, Sundaresan V (2009) Auxin-dependent patterning and gamete specification in the Arabidopsis female gametophyte. Science 324:1684–1689CrossRefPubMedGoogle Scholar
  35. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263CrossRefPubMedGoogle Scholar
  36. Palovaara J, Hallberg H, Stasolla C, Hakman I (2010) Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8/9 in developing seeds and somatic embryos of the gymnosperm Picea abies. N Phytol 188:122–135CrossRefGoogle Scholar
  37. Pang M, Woodward AW, Agarwal V, Guan X, Ha M, Ramachandran V, Chen XM, 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:R122CrossRefPubMedPubMedCentralGoogle Scholar
  38. Parry G, Estelle M (2006) Auxin receptors: a new role for F-box proteins. Curr Opin Cell Biol 18:152–156CrossRefPubMedGoogle Scholar
  39. Pelaez P, Trejo MS, Iniguez LP, Estrada-Navarrete G, Covarrubias AA, Reyes JL, Sanchez F (2012) Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing. BMC Genom 13:83CrossRefGoogle Scholar
  40. Peng H, Chun J, Ai TB, Tong YA, Zhang R, Zhao MM, Chen F, Wang SH (2012) MicroRNA profiles and their control of male gametophyte development in rice. Plant Mol Biol 80:85–102CrossRefPubMedGoogle Scholar
  41. Pennell RI, Lamb C (1997) Programmed cell death in plants. Plant Cell 9:1157–1168CrossRefPubMedPubMedCentralGoogle Scholar
  42. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Gene Dev 20:3407–3425CrossRefPubMedPubMedCentralGoogle Scholar
  43. Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49:592–606CrossRefPubMedGoogle Scholar
  44. Rodriguez RE, Mecchia MA, Debernardi JM, Schommer C, Weigel D, Palatnik JF (2010) Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development 137:103–112CrossRefPubMedPubMedCentralGoogle Scholar
  45. Schaller GE, Bishopp A, Kieber JJ (2015) The yin-yang of hormones: cytokinin and auxin interactions in plant development. Plant Cell 27:44–63CrossRefPubMedPubMedCentralGoogle Scholar
  46. Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527CrossRefPubMedGoogle Scholar
  47. Shen Y, Zhao Q, Zou J, Wang W, Gao Y, Meng J, Wang JB (2014) Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids. Plant Mol Biol 85:287–299CrossRefPubMedGoogle Scholar
  48. Song Y, Wang L, Xiong L (2009) Comprehensive expression profiling analysis of OsIAA gene family in developmental processes and in response to phytohormone and stress treatments. Planta 229:577–591CrossRefPubMedGoogle Scholar
  49. Sorin C, Declerck M, Christ A, Blein T, Ma L, Lelandais-Brière C, Njo MF, Beeckman T, Crespi M, Hartmann C (2014) A miR169 isoform regulates specific NF-YA targets and root architecture in Arabidopsis. N Phytol 202:1197–1211CrossRefGoogle Scholar
  50. Thirumurugan T, Ito Y, Kubo T, Serizawa A, Kurata N (2008) Identification, characterization and interaction of HAP family genes in rice. Mol Genet Genom 279:279–289CrossRefGoogle Scholar
  51. Tian C, Muto H, Higuchi K, Matamura T, Tatematsu K, Koshiba T, Yamamoto KT (2004) Disruption and overexpression of auxin response factor 8 gene of Arabidopsis affect hypocotyls elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition. Plant J 40:333–343CrossRefPubMedGoogle Scholar
  52. Varkonyi-Gasic E, Wu RM, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12CrossRefPubMedPubMedCentralGoogle Scholar
  53. Wang L, Gu X, Xu D, Wang W, Wang H, Zeng MH, Chang ZY, Huang H, Cui XF (2011) miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis. J Exp Bot 62:761–773CrossRefPubMedGoogle Scholar
  54. Webb MC, Gunning BE (1990) Embryo sac development in Arabidopsis thaliana. Sex Plant Reprod 3:244–256CrossRefGoogle Scholar
  55. Wu Y, Yang LY, Cao AQ, Wang JB (2015) Gene expression profiles in rice developing ovules provided evidence for the role of sporophytic tissue in female gametophyte development. PLoS One 10:e0141613CrossRefPubMedPubMedCentralGoogle Scholar
  56. Wynn AN, Rueschhoff EE, Frank RG (2011) Transcriptomic characterization of a synergistic genetic interaction during carpel margin meristem development in Arabidopsis thaliana. PLoS One 6:e26231CrossRefPubMedPubMedCentralGoogle Scholar
  57. Xie F, Jones DC, Wang Q, Sun R, Zhang B (2015) Small RNA sequencing identifies miRNA roles in ovule and fiber development. Plant Biotechnol J 13:355–369CrossRefPubMedGoogle Scholar
  58. Xu X, Bai H, Liu C, Chen E, Chen Q, Zhuang J, Shen B (2014) Genome-wide analysis of microRNAs and their target genes related to leaf senescence of rice. PLoS One 9:e114313CrossRefPubMedPubMedCentralGoogle Scholar
  59. Yi R, Zhu Z, Hu J, Qian Q, Dai J, Ding Y (2013) Identification and expression analysis of microRNAs at the grain filling stage in rice (Oryza sativa L.) via deep sequencing. PLoS One 8:e57863CrossRefPubMedPubMedCentralGoogle Scholar
  60. Zhang YC, Yu Y, Wang CY, Li ZY, Liu Q, Xu J, Liao JY, Wang XJ, Qu LH, Chen F, Xin PY, Yan CY, Chu JF, Li HQ, Chen YQ (2013) Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nat Biotechnol 31:848–852CrossRefPubMedGoogle Scholar
  61. Zhao Y, Hu Y, Dai M, Huang L, Zhou DX (2009) The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice. Plant Cell 21:736–748CrossRefPubMedPubMedCentralGoogle Scholar
  62. Zhu QH, Upadhyaya NM, Gubler F, Helliwell CA (2009) Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa). BMC Plant Biol 9:1CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Hybrid Rice, College of Life SciencesWuhan UniversityWuhanChina

Personalised recommendations