Skip to main content
SpringerLink
Log in

Over-expression of miR158 causes pollen abortion in Brassica campestris ssp. chinensis

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Key message

We identified and cloned the two precursors of miR158 and its target gene in Brassica campestris ssp. chinensis, which both had high relative expression in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility, which was caused by the degradation of pollen contents from the binucleate microspore stage. These results first suggest the role of miR158 in pollen development of Brassica campestris ssp. chinensis.

Abstract

MicroRNAs (miRNAs) play crucial roles in many important growth and development processes both in plants and animals by regulating the expression of their target genes via mRNA cleavage or translational repression. In this study, miR158, a Brassicaceae specific miRNA, was functionally characterized with regard to its role in pollen development of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Two family members of miR158 in B. campestris, namely bra-miR158a1 and bra-miR158a2, and their target gene bra027656, which encodes a pentatricopeptide repeat (PPR) containing protein, were identified. Then, qRT-PCR analysis and GUS-reporter system revealed that both bra-miR158 and its target gene had relatively high expression levels in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility and pollen germination ratio, and the degradation of pollen contents from the binucleate microspore stage was also found in those deformed pollen grains, which led to pollen shrinking and collapse in later pollen development stage. These results first shed light on the importance of miR158 in pollen development of Brassica campestris ssp. chinensis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  • Allen G, Flores-Vergara M, Krasynanski S, Kumar S, Thompson W (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat protoc 1:2320–2325

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barkan A, Small I (2014) Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol 65:415–442

    Article  CAS  PubMed  Google Scholar 

  • Barkan A, Rojas M, Fujii S, Yap A, Chong YS, Bond CS, Small I (2012) A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. Plos Genet 8:e1002910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Bhardwaj AR, Joshi G et al (2014) A Genome-Wide perspective of miRNAome in response to high temperature, salinity and drought stresses in Brassica juncea (Czern) L. PloS One 9:e92456

    Article  PubMed  PubMed Central  Google Scholar 

  • Bhatt AM, Canales C, Dickinson HG (2001) Plant meiosis: the means to 1 N. Trends Plant Sci 6:114–121

    Article  CAS  PubMed  Google Scholar 

  • Blackmore S, Barnes SH (1990) Pollen wall development in angiosperms. Microspores: evolution and ontogeny. Academic Press, London pp 173–192

    Book  Google Scholar 

  • Bonhomme S, Budar F, Lancelin D, Small I, Defrance MC, Pelletier G (1992) Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica cybrids. Mol Gen Genet 235:340–348

    Article  CAS  PubMed  Google Scholar 

  • Bushati N, Cohen SM (2007) microRNA functions. Annu Rev Cell Dev Biol 23:175–205

    Article  CAS  PubMed  Google Scholar 

  • Chambers C, Shuai B (2009) Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR. BMC Plant Bio 9:1

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Chen ZH, Bao ML et al (2011) Regulation of auxin response by miR393-targeted transport inhibitor response protein1 is involved in normal development in Arabidopsis. Plant Mol Bio 77:619–629

    Article  CAS  Google Scholar 

  • Cheng F, Mandáková T, Wu J, Xie Q, Lysak M, Wang X (2013) Deciphering the diploid ancestral genome of the mesohexaploid Brassica rapa. Plant Cell 25:1541–1554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Desloire S, Hassen G et al (2003) Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family. EMBO Rep 4:588–594

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Drakakaki G, Zabotina O, Delgado I, Robert S, Keegstra K, Raikhel N (2006) Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development. Plant Physiol 142:1480–1492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dresselhaus T, Franklin-Tong N (2013) Male–female crosstalk during pollen germination, tube growth and guidance, and double fertilization. Mol Plant 6:1018–1036

    Article  CAS  PubMed  Google Scholar 

  • Fahlgren N, Howell MD et al (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS One 2:e219

    Article  PubMed  PubMed Central  Google Scholar 

  • Fahlgren N, Jogdeo S et al (2010) MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana. Plant Cell 22:1074–1089

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fei H, Sawhney VK (2011) Ultrastructural characterization of male sterile33 (ms33) mutant in Arabidopsis affected in pollen desiccation and maturation. Can J Bot 79:118–129

    Google Scholar 

  • Grant-Downton R, Trionnaire GL et al (2009) MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana. BMC Genomics 10:643

    Article  PubMed  PubMed Central  Google Scholar 

  • Hu JY, Zhou Y et al (2014) miR824-Regulated AGAMOUS-LIKE16 contributes to flowering time repression in Arabidopsis. Plant Cell 26:2024–2037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang L, Cao J, Ye W, Liu T, Jiang L, Ye Y (2008) Transcriptional differences between the male-sterile mutant bcms and wild-type Brassica campestris ssp. chinensis reveal genes related to pollen development. Plant Bio 10:342–355

    Article  CAS  Google Scholar 

  • Jiang J, Jiang J, Yang Y, Cao J (2013) Identification of microRNAs potentially involved in male sterility of Brassica campestris ssp. chinensis using microRNA array and quantitative RT-PCR assays. Cell Mol Bio Lett 18:416–432

    CAS  Google Scholar 

  • Jiang J, Lv M, Liang Y, Ma Z, Cao J (2014) Identification of novel and conserved miRNAs involved in pollen development in Brassica campestris ssp. chinensis by high-throughput sequencing and degradome analysis. BMC Genomics 15:1

    Article  CAS  Google Scholar 

  • Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787–799

    Article  CAS  PubMed  Google Scholar 

  • Juarez MT, Kui JS, Thomas J, Heller BA, Timmermans MC (2004) microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature 428:84–88

    Article  CAS  PubMed  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  CAS  PubMed  Google Scholar 

  • Kutter C, Schöb H, Stadler M, Jr MF, Si-Ammour A (2007) MicroRNA-mediated regulation of stomatal development in Arabidopsis. Plant Cell 19:2417–2429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin S, Dong H, Zhang F, Qiu L, Wang F, Cao J, Huang L (2014) BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris. Annu Bot. doi:10.1093/aob/mct315

    Google Scholar 

  • Liu W, Xu L et al (2015) Transcriptome-wide analysis of chromium-stress responsive microRNAs to explore miRNA-mediated regulatory networks in radish (Raphanus sativus L.). Sci Rep 5

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2– ∆∆CT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Lu Y, Li C, Wang H, Chen H, Berg H, Xia Y et al (2011) AtPPR2, an Arabidopsis pentatricopeptide repeat protein, binds to plastid 23 S rRNA and plays an important role in the first mitotic division during gametogenesis and in cell proliferation during embryogenesis. Plant J 67:13–25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lurin C, Small I (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–2103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lyu ML Yu YJ et al. (2015) BcMF26a and BcMF26b are duplicated polygalacturonase genes with divergent expression patterns and functions in pollen development and pollen tube formation in Brassica campestris. PloS One 10:e0131173

    Article  PubMed  PubMed Central  Google Scholar 

  • Maher C, Stein L, Ware D (2006) Evolution of Arabidopsis microRNA families through duplication events. Genome Res 16:510–519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McCormick S (1993) Male gametophyte development. Plant Cell 5:1265

    Article  PubMed  PubMed Central  Google Scholar 

  • McCormick S (2004) Control of male gametophyte development. Plant Cell 16:S142–S153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McHale NA, Koning RE (2004) MicroRNA-directed cleavage of Nicotiana sylvestris PHAVOLUTA mRNA regulates the vascular cambium and structure of apical meristems. Plant Cell 16:1730–1740

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miao YX, Dong Y et al (2012) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:1–15

    Article  Google Scholar 

  • Moon S, Kim SR et al (2013) Rice GLYCOSYLTRANSFERASE1 encodes a glycosyltransferase essential for pollen wall formation. Plant Physiol 161:663–675

    Article  CAS  PubMed  Google Scholar 

  • Mora JRH, Rivals E, Mireau H, Budar F (2010) Sequence analysis of two alleles reveals that intra-and intergenic recombination played a role in the evolution of the radish fertility restorer (Rfo). BMC Plant Bio 10:35

    Article  Google Scholar 

  • Nogueira FT, Madi S, Chitwood DH, Juarez MT, Timmermans MC (2007) Two small regulatory RNAs establish opposing fates of a developmental axis. Genes Dev 21:750–755

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Owen HA, Makaroff CA (1995) Ultrastructure of microsporogenesis and microganetogenesis in Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae). Protoplasma 185:7–21

    Article  Google Scholar 

  • Palatnik JF, Wollmann H et al (2007) Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319. Dev Cell 13:115–125

    Article  CAS  PubMed  Google Scholar 

  • Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2007) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425

    Article  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520

    Article  CAS  PubMed  Google Scholar 

  • Ru P, Xu L, Ma H, Huang H (2006) Plant fertility defects induced by the enhanced expression of microRNA167. Cell Res 16:457–465

    Article  CAS  PubMed  Google Scholar 

  • Rubio-Somoza I, Zhou CM et al (2014) Temporal control of leaf complexity by miRNA-regulated licensing of protein complexes. Curr Biol 24:2714–2719

    Article  CAS  PubMed  Google Scholar 

  • Schmittgen TD, Lee EJ, Jiang J, Sarkar A, Yang L, Elton TS, Chen C (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44:31–38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Shi J, Cui M, Yang L, Kim Y-J, Zhang D (2015) Genetic and biochemical mechanisms of pollen wall development. Trends Plant Sci 20:741–753

    Article  CAS  PubMed  Google Scholar 

  • Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2:755–767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sun J, Zhou M, Mao Z, Li C (2012) Characterization and evolution of microRNA genes derived from repetitive elements and duplication events in plants. PloS One 7:e34092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tang G, Tang X (2013) Short Tandem Target Mimic: A long journey to the engineered molecular landmine for selective destruction/blockage of microRNAs in plants and animals. J Genet Genomics 40:291–296

    Article  CAS  PubMed  Google Scholar 

  • Tang G, Yan J, Gu Y, Qiao M, Fan R, Mao Y, Tang X (2012) Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods 58:118–125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Uyttewaal M, Arnal N et al (2008) Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by the fertility restorer locus for Ogura cytoplasmic male sterility. Plant Cell. doi:10.1105/tpc.107.057208

    PubMed  PubMed Central  Google Scholar 

  • Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Wang H et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nature Genet 43:1035–1039

    Article  CAS  PubMed  Google Scholar 

  • Wang J, Yang X, Xu H, Chi X, Zhang M, Hou X (2012) Identification and characterization of microRNAs and their target genes in Brassica oleracea. Gene 505:300–308

    Article  CAS  PubMed  Google Scholar 

  • Wei LQ, Yan LF, Wang T (2011) Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa. Genome Biol 12:1

    Article  Google Scholar 

  • Wu MF, Tian Q, Reed JW (2006) Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development 133:4211–4218

    Article  CAS  PubMed  Google Scholar 

  • Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138:2145–2154

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yeung EC, Oinam GS, Yeung SS, Harry I (2011) Anther, pollen and tapetum development in safflower, Carthamus tinctorius L. Sex Plant Reprod 24:307–317

    Article  PubMed  Google Scholar 

  • Yu X, Cao J, Ye W, Wang Y (2004) Construction of an antisense CYP86MF gene plasmid vector and production of a male-sterile Chinese cabbage transformant by the pollen-tube method. J Hortic Sci Biotechnol 79:833–839

    Article  CAS  Google Scholar 

  • Zheng B, Chen X, Mccormick S (2011) The anaphase-promoting complex is a dual integrator that regulates both MicroRNA-mediated transcriptional regulation of cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte development. Plant Cell 23:1033–1046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Program on Key Basic Research Projects (No. 2012CB113900) and the Natural Science Foundation of China (No. 31471877).

Author contributions

ZM and JJ designed all the experiments, and as the core researchers, they contributed equally to this work. ZH, TL, YY, JJ participated in this work and the manuscript modification. JC is the corresponding author. All authors read and approved the final manuscript.

Author information

Author notes

    Authors and Affiliations

    1. Lab of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, 310058, China

      Zhiming Ma, Jianxia Jiang, Ziwei Hu, Tianqi Lyu, Yang Yang & Jiashu Cao

    2. State Key Lab of Agrobiotechnology, Shenzhen Base, Shenzhen Research Institute, Chinese University of Hong Kong, Shenzhen, 518057, China

      Jingjing Jiang

    Authors
    1. Zhiming Ma
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Jianxia Jiang
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Ziwei Hu
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Tianqi Lyu
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Yang Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Jingjing Jiang
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Jiashu Cao
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Jiashu Cao.

    Ethics declarations

    Conflict of interest

    The authors declare that they have no competing interests.

    Additional information

    Zhiming Ma and Jianxia Jiang contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Supplementary material 1 (PDF 935 KB)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Ma, Z., Jiang, J., Hu, Z. et al. Over-expression of miR158 causes pollen abortion in Brassica campestris ssp. chinensis . Plant Mol Biol 93, 313–326 (2017). https://doi.org/10.1007/s11103-016-0563-7

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s11103-016-0563-7

    Keywords

    Search

    Navigation