Expression of the Floral Repressor miRNA156 is Positively Regulated by the AGAMOUS-like Proteins AGL15 and AGL18

  • Serivichyaswat, Phanu (Creative Research Initiatives, Department of Life Sciences, Korea University) ;
  • Ryu, Hak-Seung (Creative Research Initiatives, Department of Life Sciences, Korea University) ;
  • Kim, Wanhui (Creative Research Initiatives, Department of Life Sciences, Korea University) ;
  • Kim, Soonkap (Creative Research Initiatives, Department of Life Sciences, Korea University) ;
  • Chung, Kyung Sook (Creative Research Initiatives, Department of Life Sciences, Korea University) ;
  • Kim, Jae Joon (Creative Research Initiatives, Department of Life Sciences, Korea University) ;
  • Ahn, Ji Hoon (Creative Research Initiatives, Department of Life Sciences, Korea University)
  • Received : 2014.11.12
  • Accepted : 2014.12.11
  • Published : 2015.03.31


The regulation of flowering time has crucial implications for plant fitness. MicroRNA156 (miR156) represses the floral transition in Arabidopsis thaliana, but the mechanisms regulating its transcription remain unclear. Here, we show that two AGAMOUS-like proteins, AGL15 and AGL18, act as positive regulators of the expression of MIR156. Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression. Histochemical analysis further indicated that the double mutants showed a reduction in MIR156 promoter strength. The double mutants also showed reduced abundance of pri-miR156a and pri-miR156c, two of the primary transcripts from MIR156 genes. Electrophoretic mobility shift assays demonstrated that AGL15 directly associated with the CArG motifs in the MIR156a/c promoters. AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15. GFP reporter assays and bimolecular fluorescence complementation (BiFC) showed that AGL15 and AGL18 co-localize in the nucleus and confirmed their in vivo interaction. Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts. Taking these data together, we present a model for the transcriptional regulation of MIR156. In this model, AGL15 and AGL18 may form a complex along with other proteins, and bind to the CArG motifs of the promoters of MIR156 to activate the MIR156 expression.


AGAMOUS-like 15;AGAMOUS-like 18;CArG motifs;floral transition;miRNA156


Grant : KU 생명과학 창의인재양성사업단


  1. Adamczyk, B.J., Lehti-Shiu, M.D., and Fernandez, D.E. (2007). The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis. Plant J. 50, 1007-1019
  2. Borner, R., Kampmann, G., Chandler, J., Gleissner, R., Wisman, E., Apel, K., and Melzer, S. (2000). A MADS domain gene involved in the transition to flowering in Arabidopsis. Plant J. 24, 591-599
  3. Carrington, C., and Ambros, V., (2003). Role of microRNAs in plant and animal development. Science 301, 336-338
  4. Chen, X. (2004). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022-2025.
  5. Cho, H.J., Kim, J.J., Lee, J.H., Kim, W., Jung, J., Park, C., and Ahn, J.H. (2012). SHORT VEGETATIVE PHASE (SVP) protein negatively regulates miR172 transcription via direct binding to the primiR172a promoter in Arabidopsis. FEBS Lett. 586, 2332-2337
  6. Chung, Y., Kwon, S.I., and Choe, S. (2014). Antagonistic regulation of Arabidopsis growth by brassinosteroids and abiotic stresses. Mol. Cells 37, 795-803.
  7. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.
  8. Ferrandiz, C., Liljegren, S.J., and Yanofsky, M.F. (2000). Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science 289, 436-438
  9. Fernandez, D.E., Wang, C.T., Zheng, Y., Adamczyk, B.J., Singhal, R., Hall, P.K., and Perry, S.E. (2014). The MADS-domain factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, are necessary to block floral gene expression during the vegetative phase. Plant Physiol. 165, 1591-1603.
  10. Fornara, F., Montaigu, A., and Coupland, G. (2010). Snapshot: control of flowering in Arabidopsis. Cell 141, 550.e1-2.
  11. Gu, X., Wang, Y., and He, Y. (2013). Photoperiodic regulation of flowering time through periodic histone deacetylation of the florigen gene FT. PLoS Biol. 11, E1001649
  12. Hehl, R., and Bulow, L. (2014). AthaMap web tools for the analysis of transcriptional and posttranscriptional regulation of gene expression in Arabidopsis thaliana. Methods Mol. Biol. 1158, 139-156.
  13. Higo, K., Ugawa, Y., Iwamoto, M., and Higo, H. (1998). PLACE: a database of plant cis-acting regulatory DNA elements. Nucleic Acids Res. 26, 358-359
  14. Hill, K., Wang, H., and Perry, S.E. (2008). A transcriptional repression motif in the MADS factor AGL15 is involved in recruitment of histone deacetylase complex components. Plant J. 53, 172-185.
  15. Hong, S.M., Bahn, S.C., Lyu, A., Jung, H.S., and Ahn, J.H. (2010). Identification and testing of superior reference genes for a starting pool of transcript normalization in Arabidopsis. Plant Cell Physiol. 51, 1694-1606.
  16. Jack, T. (2001). Plant development going MADS. Plant Mol. Biol. 46, 515-520.
  17. Jang, Y.H., Park, H., Kim, S., Lee, J.H., Suh, M.C., Chung, Y.S., Peak, K., and Kim, J. (2009) Survey of rice proteins interacting with OsFCA and OsFY proteins which are homologous to the Arabidopsis flowering time proteins, FCA and FY. Plant Cell Physiol. 5, 1479-1492.
  18. Kutter, C., Schob, H., Stadler, M., Meins, F., and Si-Ammour, A. (2007). MicroRNA-mediated regulation of stomatal development in Arabidopsis. Plant Cell 19, 2417-2429
  19. Lee, J.H., Yoo, S.J., Park, S.H., Hwang, I., Lee, J.S., and Ahn, J.H. (2007). Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. Gene Dev. 21, 397-402.
  20. Lee, J.H., Lee, J.S., and Ahn, J.H. (2008). Ambient temperature signaling in plants: an emerging field in the regulation of flowering time. J. Plant Biol. 51, 321-326.
  21. Lee, H., Yoo, S.J., Lee, J.H., Kim, W., Yoo, S.K., Fitzgarald, H., Carrington, J.C., and Ahn, J.H. (2010). Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis. Nucleic Acids Res. 38, 3081-3093.
  22. Lee, J.H., Kim, J.J., and Ahn, J.H. (2012a). Role of SEPALLATA3 (SEP3) as a downstream gene of miR156-SPL3-FT circuitry in ambient temperature-responsive flowering. Plant Signal. Behav. 7, 1151-1154
  23. Lee, J.H., Park, S.H., and Ahn, J.H. (2012b). Functional conservation and diversification between rice OsMADS22/OsMADS55 and Arabidopsis SVP proteins. Plant Sci. 185-186, 97-104.
  24. Nesi, N., Debeaujon I., Jond C., Stewart A.J., Jenkins G.I., Caboche M., and Lepiniec, L. (2002). The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. Plant Cell 14, 2463-2479
  25. Kim, J.J., Lee, J.H., Kim, W., Jung, H.S., Huijser, P., and Ahn, J.H. (2012). The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis thaliana. Plant Physiol. 159, 461-478
  26. Perry, S.E., Nichols, K.W., and Fernandez, D.E. (1996). The MADS domain protein AGL15 localizes to the nucleus during early stages of seed development. Plant Cell 8, 1977-1989
  27. Pinyopich, A., Ditta, G.S., Savidge, B., Liljegren, S.J., Baumann, E., Wisman, E., and Yanofsky, M.F. (2003). Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424, 85-88
  28. Riechmann, J.L., Krizek, B.A., and Meyerowitz, E.M. (1996). Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Proc. Natl. Acad. Sci. USA 93, 4793-4798
  29. Rubio-Somoza, I., and Weigel, D. (2013). Coordination of flower maturation by a regulatory circuit of three microRNAs. PLoS Genet. 9, e1003374
  30. Shore, P., and Sharrocks, A.D. (1995). The MADS-box family of transcription factors. Eur. J. Biochem. 229, 1-13.
  31. Tang, W., and Perry, S.E. (2003). Binding site selection for the plant MADS domain protein AGL15: an in vitro and in vivo study. J. Biol Chem. 278, 28154-28159
  32. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949-956.
  33. Walter, M., Chaban, C., Schuutze, K., Batistic, O., Weckermann, K., Nake, C., Blazervic, D., Grefen, C., Schumacher, K., Oecking, C., et al. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428-438
  34. Wang, H., Tang, W., Zhu, C., and Perry, S.E. (2002). A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos. Plant J. 32, 831-843.
  35. Wang, J., Czech, B., and Weigel, D. (2009). MiR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell 138, 738-749
  36. West, A.G., Shore, P., and Sharrocks, A.D. (1997). DNA binding by MADS-Box transcription factors: a molecular mechanism for differential DNA bending. Mol. Cell. Biol. 17, 2876-2887
  37. Wu, G., and Poethig, R.S. (2006). Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133, 3539-3547
  38. Xie, Z., Allen E.A., Fahlgren N., Calamar, A., Givan, S.A., and Carrington J.C. (2005). Expression of Arabidopsis MIRNA genes. Plant Physiol. 138, 2145-2154.
  39. Yamaguchi, A., Wu, M., Yang, L., Wu, G., Poethig, R.S., and Wagner, D. (2009). The MicroRNA-regulated SBP-box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1. Dev. Cell 17, 268-278
  40. Yoo, S.D., Cho, Y.H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565-1572.
  41. Yoo, S. K., Wu, X., Lee, J.S., and Ahn, J.H. (2011). AGAMOUSLIKE 6 is a floral promoter that negatively regulates the FLC/MAF clade genes and positively regulates FT in Arabidopsis. Plant J. 65, 62-76.
  42. Zhang, H. and Forde, B.G. (1998). An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279, 407-409
  43. Zhang, Y., Cao G., Qu, L., and Gu, H. (2009). Characterization of Arabidopsis MYB transcription factor gene AtMYB17 and its possible regulation by LEAFY and AGL15. J. Genet. Genomics 36, 99-107

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