Plant Molecular Biology

, Volume 61, Issue 4–5, pp 781–793 | Cite as

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

  • Sizolwenkosi Mlotshwa
  • Zhiyong Yang
  • YunJu Kim
  • Xuemei Chen


Floral patterning and morphogenesis are controlled by many transcription factors including floral homeotic proteins, by which floral organ identity is determined. Recent studies have uncovered widespread regulation of transcription factors by microRNAs (miRNAs), ~21-nucleotide non-coding RNAs that regulate protein-coding RNAs through transcript cleavage and/or translational inhibition. The regulation of the floral homeotic gene APETALA2 (AP2) by miR172 is crucial for normal Arabidopsis flower development and is likely to be conserved across plant species. Here we probe the activity of the AP2/miR172 regulatory circuit in a heterologous Solanaceae species, Nicotiana benthamiana. We generated transgenic N. benthamiana lines expressing Arabidopsis wild type AP2 (35S::AP2), miR172-resistant AP2 mutant (35S::AP2m3) and MIR172a-1 (35S::MIR172) under the control of the cauliflower mosaic virus 35S promoter. 35S::AP2m3 plants accumulated high levels of AP2 mRNA and protein and exhibited floral patterning defects that included proliferation of numerous petals, stamens and carpels indicating loss of floral determinacy. On the other hand, nearly all 35S::AP2 plants accumulated barely detectable levels of AP2 mRNA or protein and were essentially non-phenotypic. Overall, the data indicated that expression of the wild type Arabidopsis AP2 transgene was repressed at the mRNA level by an endogenous N. benthamiana miR172 homologue that could be detected using Arabidopsis miR172 probe. Interestingly, 35S::MIR172 plants had sepal-to-petal transformations and/or more sepals and petals, suggesting interference with N. benthamiana normal floral homeotic gene function in perianth organs. Our studies uncover the potential utility of the Arabidopsis AP2/miR172 system as a tool for manipulation of floral architecture and flowering time in non-model plants.


APETALA2 Arabidopsis microRNA miR172 Nicotiana benthamiana 







cauliflower mosaic virus






high molecular weight RNA


low molecular weight RNA






Nicotiana tabacum AGAMOUS


Nicotiana benthamiana AGAMOUS


Nicotiana benthamiana APETALA2


Nicotiana benthamiana DEFICIENS


Nicotiana benthamiana GLOBOSA


Nicotiana benthamiana WUSCHEL


ribulose bisphosphate carboxylase






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  1. Achard, P, Herr, A, Baulcombe, DC, Harberd, NP 2004Modulation of floral development by a gibberellin-regulated microRNADevelopment3133573365CrossRefGoogle Scholar
  2. Aukerman, MJ, Sakai, H 2003Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genesPlant Cell1527302741PubMedCrossRefGoogle Scholar
  3. Axtell, MJ, Bartel, DP 2005Antiquity of microRNAs and their targets in land plantsPlant Cell1716581673PubMedCrossRefGoogle Scholar
  4. Baker, CC, Sieber, P, Wellmer, F, Meyerowitz, EM 2005The early extra petals1 mutant uncovers a role for microRNA miR164c in regulating petal number in Arabidopsis Curr Biol15303315PubMedCrossRefGoogle Scholar
  5. Bartel, B, Bartel, DP 2003MicroRNAs: at the root of plant development?Plant Physiol132709717PubMedCrossRefGoogle Scholar
  6. Bartel, DP 2004MicroRNAs: genomics, biogenesis, mechanism and functionCell116281297PubMedCrossRefGoogle Scholar
  7. Baurle, I, Laux, T 2005Regulation of WUSCHEL transcription in the stem cell niche of the Arabidopsis shoot meristemPlant Cell1722712280PubMedCrossRefGoogle Scholar
  8. Bomblies, K, Dagenais, N, Weigel, D 1999Redundant enhancers mediate transcriptional repression of AGAMOUS by APETALA2 Dev Biol216260264PubMedCrossRefGoogle Scholar
  9. Bowman, JL, Smyth, DR, Meyerowitz, EM 1991Genetic interactions among floral homeotic genes of Arabidopsis Development112120PubMedGoogle Scholar
  10. Carrington, JC, Ambros, V 2003Role of microRNAs in plant and animal developmentScience301336338PubMedCrossRefGoogle Scholar
  11. Chen, X 2004A microRNA as a translational repressor of APETALA2 in Arabidopsis flower developmentScience30320222025PubMedCrossRefGoogle Scholar
  12. Chen, X 2005MicroRNA biogenesis and function in plantsFEBS Lett57959235931PubMedCrossRefGoogle Scholar
  13. Coen, ES, Meyerowitz, EM 1991The war of the whorls: genetic interactions controlling flower developmentNature3533137PubMedCrossRefGoogle Scholar
  14. Drews, GN, Bowman, JL, Meyerowitz, EM 1991Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 productCell659911002PubMedCrossRefGoogle Scholar
  15. Dugas, DV, Bartel, B 2004MicroRNA regulation of gene expression in plantsCurr Opin Plant Biol7512520PubMedCrossRefGoogle Scholar
  16. Gutierrez-Cortines, ME, Davies, B 2000Beyond the ABCs: ternary complex formation in the control of floral organ identityTrends Plant Sci5471476PubMedCrossRefGoogle Scholar
  17. Halfter, U, Ali, N, Stockhaus, J, Ren, L, Chua, N-H 1994Ectopic expression of a single homeotic gene, the Petunia gene green petal, is sufficient to convert sepals to petaloid organsEMBO J1314431449PubMedGoogle Scholar
  18. He, L, Hannon, GJ 2004MicroRNAs: small RNAs with a big role in gene regulationNat Rev Genet5522531PubMedCrossRefGoogle Scholar
  19. Jack, T 2004Molecular and genetic mechanisms of floral controlPlant Cell16S117PubMedCrossRefGoogle Scholar
  20. Kidner, CA, Martienssen, RA 2005The developmental role of microRNA in plantsCurr Opin Plant Biol83844PubMedCrossRefGoogle Scholar
  21. Kieffer, M, Stern, Y, Cook, H, Clerici, E, Maulbetsch, C, Laux, T, Davies, B 2006Analysis of the transcription factor WUSCHEL and its functional homologue in Antirrhinum reveals a potential mechanism for their roles in meristem maintenancePlant Cell18560573PubMedCrossRefGoogle Scholar
  22. Krizek, BA, Fletcher, JC 2005Molecular mechanism of flower development: an armchair guideNat Rev Genet6688698PubMedCrossRefGoogle Scholar
  23. Krizek, BA, Meyerowitz, EM 1996The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity functionDevelopment1221122PubMedGoogle Scholar
  24. Krizek, BA, Prost, V, Macias, A 2000 AINTEGUMENTA promotes petal identity and acts as a negative regulator of AGAMOUS Plant Cell1213571366PubMedCrossRefGoogle Scholar
  25. Kunst, L, Klenz, JE, Martinez-Zapater, J, Haughn, GW 1989 AP2 gene determines the identity of perianth organs in flowers of Arabidopsis thaliana Plant Cell111951208PubMedCrossRefGoogle Scholar
  26. Lauter, N, Kampani, A, Carlson, S, Goebel, M, Moose, SP 2005microRNA172 down-regulates glossy15 to promote vegetative phase change in maizeProc Natl Acad Sci USA10294129417PubMedCrossRefGoogle Scholar
  27. Laux, T, Mayer, KF, Berger, J, Jurgens, G 1996The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis Development1228796PubMedGoogle Scholar
  28. Lenhard, M, Bohnert, A, Jurgens, G, Laux, T 2001Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS Cell105805814PubMedCrossRefGoogle Scholar
  29. Liu, Y, Nakayama, N, Schiff, M, Litt, A, Irish, VF, Dinesh-Kumar, SP 2004Virus induced gene silencing of a DEFICIENS ortholog in Nicotiana benthamiana Plant Mol Biol54701711PubMedCrossRefGoogle Scholar
  30. Lohmann, JU, Weigel, D 2002Building beauty: the genetic control of floral patterningDev Cell2135142PubMedCrossRefGoogle Scholar
  31. Lohmann, JU, Hong, RL, Hobe, M, Busch, MA, Parcy, F, Simon, R, Weigel, D 2001A molecular link between stem cell regulation and floral patterning in Arabidopsis Cell105793803PubMedCrossRefGoogle Scholar
  32. Maes, T, Steene, N, Zethof, J, Karimi, M, D’Hauw, M, Mares, G, Montagu, M, Gerats, T 2001Petunia AP2-like genes and their role in flower and seed developmentPlant Cell13229244PubMedCrossRefGoogle Scholar
  33. Mallory, AC, Vaucheret, H 2004MicroRNAs: something important between the genesCurr Opin Plant Biol7120125PubMedCrossRefGoogle Scholar
  34. Mallory, AC, Dugas, DV, Bartel, DP, Bartel, B 2004aMicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organsCurr Biol1410351046CrossRefGoogle Scholar
  35. Mallory, AC, Reinhart, BJ, Jones-Rhoades, MW, Tang, G, Zamore, PD, Barton, MK, Bartel, DP 2004bMicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ regionEMBO J2333563364CrossRefGoogle Scholar
  36. Meyerowitz, EM, Bowman, JL, Brockman, LL, Drews, GN, Jack, T, Sieburth, LE, Weigel, D 1991A genetic and molecular model for flower development in Arabidopsis thalianaDev Suppl1157167PubMedGoogle Scholar
  37. Millar, AA, Gubler, F 2005The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther developmentPlant Cell17705721PubMedCrossRefGoogle Scholar
  38. Mizukami, Y, Ma, H 1997Determination of Arabidopsis floral meristem identity by AGAMOUS Plant Cell9393408PubMedCrossRefGoogle Scholar
  39. Mlotshwa, S 2000The helper component-proteinase of Cowpea aphid-borne mosaic virusWageningen University and Research CentreWageningen, The NetherlandsPh.D. ThesisGoogle Scholar
  40. Mlotshwa, S, Verver, J, Sithole-Niang, I, Prins, M, Kammen, AB, Wellink, J 2002Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistanceVirus Genes254557PubMedCrossRefGoogle Scholar
  41. Parizotto, EA, Dunoyer, P, Rahm, N, Himber, C, Voinnet, O 2004In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNAGenes Dev1822372242PubMedCrossRefGoogle Scholar
  42. Schmid, M, Uhlenhaut, NH, Godard, F, Demar, M, Bressan, R, Weigel, D, Lohmann, JU 2003Dissection of floral induction pathways using global expression analysisDevelopment13060016012PubMedCrossRefGoogle Scholar
  43. Schoof, H, Lenhard, M, Haecker, A, Mayer, KF, Jurgens, G, Laux, T 2000The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genesCell100635644PubMedCrossRefGoogle Scholar
  44. Schwab, R, Palatnik, JF, Riester, M, Schommer, C, Schmid, M, Weigel, D 2005Specific effects of microRNAs on the plant transcriptomeDev Cell8517527PubMedCrossRefGoogle Scholar
  45. Valencia-Sanchez, MA, Liu, J, Hannon, GJ, Parker, R 2006Control of translation and mRNA degradation by miRNAs and siRNAsGenes Dev20515524PubMedCrossRefGoogle Scholar
  46. Wurschum, T, Gross-Hardt, R, Laux, T 2006 APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristemPlant Cell18295307PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Sizolwenkosi Mlotshwa
    • 1
  • Zhiyong Yang
    • 1
  • YunJu Kim
    • 1
  • Xuemei Chen
    • 1
  1. 1.Department of Botany & Plant SciencesUniversity of CaliforniaCalifornia, RiversideUSA

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