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Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis

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Abstract

Somatic embryo (SE) regeneration is an ideal experimental system to realize rapid propagation of excellent clones and genetic improvement for perennial gymnosperms. In the present study, genes encoding the miRNA166 precursor were identified and LamiR166a was successfully transformed into the gymnosperm Larix leptolepis (L. leptolepis) and five LamiR166a over-expressed embryonic cell lines were screened out as stable embryo masses. As expected, the targets of miR166a, LaHDZ31-34, were all down-regulated in transgenic lines according to qRT-PCR results. The results showed that the percentage of normal SEs with 4–7 cotyledons was 77.0 % in wild type (WT) lines, but was reduced to 60.3 % in the pSuper::MIR166a lines with “cup-shaped” embryos comprised 7.0 % of WT and 20.7 % of transgenic embryos. Microscopic observation further showed that the intermediate region surrounded by the cotyledons was larger than in the control, with no upward bulge of the shoot apical meristem (SAM). The expression pattern of the two meristem marker genes CLAVATA (CLV) and WUSCHEL-related homeobox (WOX) were investigated. The results showed that the expression levels of WOX were three times higher in transgenic lines than in WT samples, which suggest that miR166a may indirectly regulate SAM development by directly affecting WOX expression. Besides, overexpression of LamiR166a clearly increased the rooting rate and promoted lateral root formation in L. leptolepis seedlings. These results may provide new insights into the regulatory role of miR166 in gymnosperms, and also new applications for forestry production in practice.

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References

  • Brand U, Fletcher JC, Hobe M, Meyerowitz EM, Simon R (2000) Dependence of stem cell fate in arabidopsis on a feedback loop regulated by CLV3 activity. Science 289(5479):617–619

    Article  CAS  PubMed  Google Scholar 

  • Breuninger H, Rikirsch E, Hermann M, Ueda M, Laux T (2008) Differential expression of WOX, genes mediates apical-basal axis formation in the arabidopsis, embryo. Dev Cell 14(6):867–876

    Article  CAS  PubMed  Google Scholar 

  • Carlsbecker A, Lee JY, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O, Moreno-Risueno MA, Vaten A, Thitamadee S, Campilho A, Sebastian J, Bowman JL, Helariutta Y, Benfey PN (2010) Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465:316–321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chou H, Zhu Y, Ma Y, Berkowitz GA (2016) The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires ca2+ as a secondary cytosolic messenger. Plant J 85(4):494–506

    Article  CAS  PubMed  Google Scholar 

  • Cote CL, Boileau F, Roy V, Ouellet M, Levasseur C, Morency MJ, Cooke JE, Seguin A, MacKay JJ (2010) Gene family structure, expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees. BMC Plant Biol 10:273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Den Herder G, Van Isterdael G, Beeckman T, De Smet I (2010) The roots of a new green revolution. Trends Plant Sci 15:600–607

    Article  CAS  PubMed  Google Scholar 

  • Deveaux Y, Toffano-Nioche C, Claisse G, Thareau V, Morin H, Laufs P et al (2007) Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis. BMC Evol Biol 8(6):665–680

    Google Scholar 

  • Dolzblasz A, Nardmann J, Clerici E, Causier B, van der Graaff E, Chen J et al (2016) Stem cell regulation by arabidopsis WOX genes. Mol Plant 9(7):1028–1039

    Article  CAS  PubMed  Google Scholar 

  • Ehrenreich IM, Purugganan MD (2008) Sequence variation of micrornas and their binding sites in arabidopsis. Plant Physiol 146(4):1974–1982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Floyd SK, Zalewski CS, Bowman JL (2006) Evolution of class III homeodomain-leucine zipper genes in streptophytes. Genetics 173:373–388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fu X, Harberd NP (2003) Auxin promotes Arabidopsis root growth by modulating gibberellin response. Nature 421(6924):740–743

    Article  CAS  PubMed  Google Scholar 

  • Gou J, Strauss SH et al (2010) Gibberellins regulate lateral root formation in populus through interactions with auxin and other hormones. Plant Cell 22(3):623–639

    Article  CAS  PubMed  Google Scholar 

  • Griffiths J, Murase K et al (2006) Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis. Plant Cell 18(12):3399–3414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jansen L, Roberts I, De Rycke R, Beeckman T (2012) Phloem-associated auxin response maxima determine radial positioning of lateral roots in maize. Philos Trans R Soc Lond B Biol Sci 367:1525–1533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jia X, Ding N, Fan W, Yan J, Gu Y, Tang X, Li R, Tang G (2015) Functional plasticity of miR165/166 in plant development revealed by small tandem target mimic. Plant Sci 233:11–21

    Article  CAS  PubMed  Google Scholar 

  • Jung J, Park C (2007) MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis. Planta 225:1327–1338

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Lavenus J, Goh T, Roberts I, Guyomarc’H S, Lucas M, De Smet I, Fukaki H, Beeckman T, Bennett M, Laplaze L (2013) Lateral root development in Arabidopsis: fifty shades of auxin. Trends Plant Sci 18:450–458

    Article  CAS  PubMed  Google Scholar 

  • Li S, Li W, Han S, Yang W, Qi L (2013a) Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos. Gene 522:177–183

  • Li W, Zhang S, Han S, Wu T, Zhang J, Qi L (2013b) Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr. Plant Cell Tissue Organ Cult (PCTOC) 113(1):131–136

  • Liu B, Wang L, Zhang J, Li J, Zheng H, Chen J et al (2014) Wuschel-related homeobox, genes in populus tomentosa: diversified expression patterns and a functional similarity in adventitious root formation. BMC Genom 15(1):1–14

    Article  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 

  • Miyashima S, Koi S, Hashimoto T, Nakajima K (2011) Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate multiple differentiation status in the Arabidopsis root. Development 138:2303–2313

    Article  CAS  PubMed  Google Scholar 

  • Nicoll BC, Ray D (1996) Adaptive growth of tree root systems in response to wind action and site conditions. Tree Physiol 16:891–898

    Article  PubMed  Google Scholar 

  • Niu S, Li Z, Yuan H, Fang P, Chen X, Li W (2013) Proper gibberellin localization in vascular tissue is required to regulate adventitious root development in tobacco. J Exp Bot 64:3411–3424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nozawa M, Miura S, Nei M (2012) Origins and evolution of microRNA genes in plant species. Genome Biol Evol 4:230–239

    Article  PubMed  PubMed Central  Google Scholar 

  • Ochando I, Jover-Gil S, Ripoll JJ, Candela H, Vera A, Ponce MR, Martinez-Laborda A, Micol JL (2006) Mutations in the microRNA complementarity site of the INCURVATA4 gene perturb meristem function and adaxialize lateral organs in Arabidopsis. Plant Physiol 141:607–619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Osipova MA, Mortier V, Demchenko KN, Tsyganov VE, Tikhonovich IA, Lutova LA, Dolgikh EA, Goormachtig S (2012) Wuschel-related homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation. Plant Physiol 158:1329–1341

    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 

  • Sakaguchi J, Watanabe Y (2012) miR165/166 and the development of land plants. Dev Growth Differ 54:93–99

    Article  CAS  PubMed  Google Scholar 

  • Schoof H, Lenhard M, Haecker A, Mayer KF, Jurgens G, Laux T (2000) The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100:635–644

    Article  CAS  PubMed  Google Scholar 

  • Singh A, Singh S, Panigrahi KCS, Reski R, Sarkar AK (2014) Balanced activity of microRNA166/165 and its target transcripts from the class III homeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana. Plant Cell Rep 33:945–953

    Article  CAS  PubMed  Google Scholar 

  • Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43

    Article  CAS  PubMed  Google Scholar 

  • Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17:49–63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ubeda-Tomas S, Swarup R et al (2008) Root growth in Arabidopsis requires gibberellin/DELLA signalling in the endodermis. Nat Cell Biol 10(5):625–628

    Article  CAS  PubMed  Google Scholar 

  • Vandenbussche M, Horstman A, Zethof J, Koes R, Rijpkema AS, Gerats T (2009) Differential recruitment of WOX transcription factors for lateral development and organ fusion in petunia and Arabidopsis. Plant Cell 21(8):2269–2283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Yang H, Shi Y, Liu J, Guo L, Zhang X, Yang S (2010) A mutant CHS3 protein with TIR-NB-LRR-LIM domains modulates growth, cell death and freezing tolerance in a temperature-dependent manner in Arabidopsis. Plant J 63:283–296

    Article  CAS  PubMed  Google Scholar 

  • Zhang J, Zhang S, Han S, Wu T, Li X, Li W, Qi L (2012a) Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta 236:647–657

  • Zhang S, Zhou J, Han S, Yang W, Li W, Wei H, Li X, Qi L (2010) Four abiotic stress-induced miRNA families differentially regulated in the embryogenic and non-embryogenic callus tissues of Larix leptolepis. Biochem Biophys Res Commun 398:355–360

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Zhang S, Han S, Li X, Qi L (2012b) Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis). Plant Cell Rep 31:1637–1657

  • Zhang JH, Zhang SG, Li SG, Han SY, Li WF, Li XM et al (2014a) Regulation of synchronism by abscisic-acid-responsive small noncoding rnas during somatic embryogenesis in larch (Larix leptolepis). Plant Cell Tissue Organ Cult 116(116):361–370

  • Zhang LF, Li WF, Xu HY, Qi LW, Han SY (2014b) Cloning and characterization of four differentially expressed cdnas encoding nfya homologs involved in responses to aba during somatic embryogenesis in Japanese larch (Larix leptolepis). Plant Cell Tissue Organ Cult 117(2):293–304

  • Zhou Y, Honda M, Zhu H, Zhang Z, Guo X, Li T, Li Z, Peng X, Nakajima K, Duan L, Zhang X (2015) Spatiotemporal sequestration of miR165/166 by Arabidopsis Argonaute 10 promotes shoot apical meristem maintenance. Cell Reports 10:1819–1827

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Zhu T (2015) Regulation of embryo development in Norway spruce by WOX transcription factors. Dissertation, Acta Universitatis Agriculturae Sueciae

  • Zhu H, Hu F, Wang R, Zhou X, Sze SH, Liou LW, Barefoot A, Dickman M, Zhang X (2011) Arabidopsis Argonaute 10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145:242–256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31330017) and the National High Technology Research and Development Program of China (2013AA102704).

Author contributions

Shui-Gen Li conceived, designed and carried out the study. Zhe-Xin Li and Li-feng Zhang together carried out the study and Zhe-Xin Li wrote the manuscript. Li-Wang Qi provided important suggestions on the experimental design and analyses. Wan-Feng Li helped to modify the manuscript. Su-ying Han provided genetic transformation guidance. Wen-hua Yang, Ya-li Liu and Yan-ru Fan offered varying degrees of help during the experimental operation. All authors read and approved the manuscript.

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    Authors and Affiliations

    1. State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, People’s Republic of China

      Zhe-Xin Li, Shui-Gen Li, Li-feng Zhang, Wan-Feng Li, Hai-yan Xu, Wen-hua Yang, Ya-li Liu & Li-Wang Qi

    2. Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, 100091, People’s Republic of China

      Su-ying Han & Yan-ru Fan

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    Zhe-Xin Li, Shui-Gen Li and Li-feng Zhang have the equally contributed to this work.

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    Li, ZX., Li, SG., Zhang, Lf. et al. Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis . Plant Cell Tiss Organ Cult 127, 461–473 (2016). https://doi.org/10.1007/s11240-016-1071-9

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