Molecular Biotechnology

, Volume 61, Issue 1, pp 20–31 | Cite as

The Constitutive Expression of a Chrysanthemum ERF Transcription Factor Influences Flowering Time in Arabidopsis thaliana

  • Xiaojuan Xing
  • Jiafu Jiang
  • Yaoyao Huang
  • Zixin Zhang
  • Aiping Song
  • Lian Ding
  • Haibing Wang
  • Jianjun Yao
  • Sumei Chen
  • Fadi Chen
  • Weimin Fang
Original Paper


AP2/ERF transcription factors (TFs) represent valuable targets for the genetic manipulation of crop plants, as they participate in the control of metabolism, growth and development, as well as in the plants’ response to environmental stimuli. Here, an ERF TF encoded by the chrysanthemum (Chrysanthemum morifolium) genome, designated CmERF110, was cloned and functionally characterized. The predicted CmERF110 polypeptide included a conserved DNA-binding AP2/ERF domain. A transient expression experiment revealed that the protein was deposited in the nucleus, and a transactivation experiment in yeast suggested that it had no transcriptional activity. The gene was transcribed in the chrysanthemum root, stem and leaf, with its transcript level following a circadian rhythm under both long and short days. The effect of constitutively expressing the gene in Arabidopsis thaliana was to accelerate flowering. Transcriptional profiling implied that its effect on floral initiation operated through the photoperiod pathway.


CmERF110 Flowering Chrysanthemum Photoperiod 







Apetala2/ethylene responsive factor


Apetala2/ethylene-responsive element-binding factors




Constans-Like 1


Dehydration responsive element-binding


Ethylene-responsive factor


Ethylene-responsive element-binding proteins


Flowering locus T




Forever young flower




Related to ABI3/VP1


Suppressor of over-expression of CO 1


Squamosa promoter binding like


Short vegetative phase


Wild type



This work was supported by funding from the Independent Innovation of Agricultural Sciences in Jiangsu Province CX(16)1025 and the Seed Industry Development Project of Shanghai.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Riechmann, J. L., & Ratcliffe, O. J. (2000). A genomic perspective on plant transcription factors. Current Opinion in Plant Biology, 3(5), 423–434.CrossRefGoogle Scholar
  2. 2.
    Riechmann, J. L. (2002). Transcriptional regulation: A genomic overview. Arabidopsis Book, 1, e0085.CrossRefGoogle Scholar
  3. 3.
    Gong, W., Shen, Y. P., Ma, L. G., Pan, Y., Du, Y. L., Wang, D. H., et al. (2004). Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiology, 135(2), 773–782.CrossRefGoogle Scholar
  4. 4.
    Rao, G., Sui, J., Zeng, Y., He, C., & Zhang, J. (2015). Genome-wide analysis of the AP2/ERF gene family in Salix arbutifolia. Plant Molecular Biology Reporter, 5(1), 132–137.Google Scholar
  5. 5.
    Licausi, F., Ohmetakagi, M., & Perata, P. (2013). APETALA2/ethylene responsive factor (AP2/ERF) transcription factors: Mediators of stress responses and developmental programs. New Phytologist, 199(3), 639–649.CrossRefGoogle Scholar
  6. 6.
    Sears, M. T., Zhang, H., Rushton, P. J., Wu, M., Han, S., Spano, A. J., et al. (2014). NtERF32: A non-NIC2 locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco. Plant Molecular Biology, 84(1–2), 49–66.CrossRefGoogle Scholar
  7. 7.
    Zhang, G., Chen, M., Chen, X., Xu, Z., Guan, S., Li, L. C., et al. (2008). Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.). Journal of Experimental Botany, 59(15), 4095–4107.CrossRefGoogle Scholar
  8. 8.
    Mizoi, J., Shinozaki, K., & Yamaguchi-Shinozaki, K. (2012). AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta, 1819(2), 86–96.CrossRefGoogle Scholar
  9. 9.
    Sakuma, Y., Liu, Q., Dubouzet, J. G., Abe, H., Shinozaki, K., & Yamaguchi-Shinozaki, K. (2002). DNA-Binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications, 290(3), 998–1009.CrossRefGoogle Scholar
  10. 10.
    Song, C. P., Agarwal, M., Ohta, M., Guo, Y., Halfter, U., Wang, P., et al. (2005). Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses. The Plant Cell, 17(8), 2384–2396.CrossRefGoogle Scholar
  11. 11.
    Zhu, L., Liu, D., Li, Y., & Li, N. (2013). Functional phosphoproteomic analysis reveals that a serine-62-phosphorylated isoform of ethylene response factor110 is involved in Arabidopsis bolting. Plant Physiology, 161(2), 904–917.CrossRefGoogle Scholar
  12. 12.
    Mehrnia, M., Balazadeh, S., Zanor, M. I., & Mueller-Roeber, B. (2013). EBE, an AP2/ERF transcription factor highly expressed in proliferating cells, affects shoot architecture in Arabidopsis. Plant Physiology, 162(2), 842–857.CrossRefGoogle Scholar
  13. 13.
    Tuan, P. A., Bai, S., Saito, T., Imai, T., Ito, A., & Moriguchi, T. (2016). Involvement of EARLY BUD-BREAK, an AP2/ERF transcription factor gene, in bud break in Japanese pear (Pyrus pyrifolia Nakai) lateral flower buds: Expression, histone modifications and possible target genes. Plant Cell Physiology, 57(5), 1038–1047.CrossRefGoogle Scholar
  14. 14.
    Li, A., Yu, X., Cao, B. B., Peng, L. X., Gao, Y., Feng, T., et al. (2017). LkAP2L2, an AP2/ERF transcription factor gene of Larix kaempferi, with pleiotropic roles in plant branch and seed development. Russian Journal of Genetics, 53(12), 1335–1342.CrossRefGoogle Scholar
  15. 15.
    Zhang, G., Chen, M., Li, L., Xu, Z., Chen, X., Guo, J., et al. (2009). Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. Journal of Experimental Botany, 60(13), 3781–3796.CrossRefGoogle Scholar
  16. 16.
    Licausi, F., Kosmacz, M., Weits, D. A., Giuntoli, B., Giorgi, F. M., Voesenek, L. A. C. J., et al. (2011). Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature, 479(7373), 419–422.CrossRefGoogle Scholar
  17. 17.
    Chung, M. Y., Vrebalov, J., Alba, R., Lee, J., Mcquinn, R., Chung, J. D., et al. (2010). A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. The Plant Journal, 64(6), 936–947.CrossRefGoogle Scholar
  18. 18.
    Zhang, Z., Yao, W., Dong, N., Liang, H., Liu, H., & Huang, R. (2007). A novel ERF transcription activator in wheat and its induction kinetics after pathogen and hormone treatments. Journal of Experimental Botany, 58(11), 2993–3003.CrossRefGoogle Scholar
  19. 19.
    Hu, L., & Liu, S. (2011). Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers. Genetics and Molecular Biology, 34(4), 624–633.CrossRefGoogle Scholar
  20. 20.
    Xu, Y. C., Hou, X. L., Xu, W. W., Shen, L. L., Lv, S. W., Zhang, S. L., et al. (2016). Isolation and characterization of an ERF-B3 gene associated with flower abnormalities in non-heading Chinese cabbage. Journal of Integrative Agriculture, 15(3), 528–536.CrossRefGoogle Scholar
  21. 21.
    Liu, J., Li, J., Wang, H., Fu, Z., Liu, J., & Yu, Y. (2011). Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments. Journal of Experimental Botany, 62(2), 825–840.CrossRefGoogle Scholar
  22. 22.
    Nakano, T., Fujisawa, M., Shima, Y., & Ito, Y. (2014). The AP2/ERF transcription factor SlERF52 functions in flower pedicel abscission in tomato. Journal of Experimental Botany, 65(12), 3111–3119.CrossRefGoogle Scholar
  23. 23.
    Shu, Y. (2010). Phosphorylated ERF110 regulates Arabidopsis inflorescence number and etiolated hypocotyl length. Phosphoprotein Phosphatases.Google Scholar
  24. 24.
    Jung, C., & Müller, A. E. (2009). Flowering time control and applications in plant breeding. Trends in Plant Science, 14(10), 563–573.CrossRefGoogle Scholar
  25. 25.
    Wang, R., Albani, M. C., Vincent, C., Bergonzi, S., Luan, M., Bai, Y., et al. (2011). Aa TFL1 confers an age-dependent response to vernalization in perennial Arabis alpina. The Plant Cell, 23(4), 1307–1321.CrossRefGoogle Scholar
  26. 26.
    Zhu, Y. D., Jin, Y. S., Wei, S., Li, H., & Zhang, W. (2012). Functional analysis of the isopentenyltransferase gene MdIPT3a from apple (Malus pumila Mill.). Journal of Horticultural Science & Biotechnology, 87(5), 478–484.CrossRefGoogle Scholar
  27. 27.
    Li, W., Ahn, I. P., Ning, Y., Park, C. H., Zeng, L., Whitehill, J. G., et al. (2012). The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis. Plant Physiology, 159(1), 239–250.CrossRefGoogle Scholar
  28. 28.
    Guo, S., Dai, S., Singh, P. K., Wang, H., Wang, Y., Tan, J., et al. (2018). A membrane-bound NAC-like transcription factor OsNTL5 represses the flowering in Oryza sativa. Frontiers in Plant Science, 9, 555.CrossRefGoogle Scholar
  29. 29.
    Zheng, T., Bo, H., Yang, Y., Li, Q., Ma, N., Ma, C., et al. (2009). Overexpression of two chrysanthemum DgDREB1 group genes causing delayed flowering or dwarfism in Arabidopsis. Plant Molecular Biology, 71(1–2), 115–129.CrossRefGoogle Scholar
  30. 30.
    Oda, A., Narumi, T., Li, T., Kando, T., Higuchi, Y., Sumitomo, K., et al. (2012). CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums. Journal of Experimental Botany, 63(3), 1461–1477.CrossRefGoogle Scholar
  31. 31.
    Sun, J., Cao, P., Wang, L., Chen, S., Chen, F., & Jiang, J. (2018). The loss of a single residue from CmFTL3 leads to the failure of florigen to flower. Plant Science, 276, 99–104.CrossRefGoogle Scholar
  32. 32.
    Mao, Y., Sun, J., Cao, P., Zhang, R., Fu, Q., Chen, S., et al. (2016). Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium. Horticulture Research, 3, 16058.CrossRefGoogle Scholar
  33. 33.
    Sun, J., Wang, H., Ren, L., Chen, S., Chen, F., & Jiang, J. (2017). CmFTL2 is involved in the photoperiod- and sucrose-mediated control of flowering time in chrysanthemum. Horticulture Research, 4, 17001.CrossRefGoogle Scholar
  34. 34.
    Higuchi, Y., Narumi, T., Oda, A., Nakano, Y., Sumitomo, K., Fukai, S., et al. (2013). The gated induction system of a systemic floral inhibitor, antiflorigen, determines obligate short-day flowering in chrysanthemums. Proc Natl Acad Sci USA, 110(42), 17137–17142.CrossRefGoogle Scholar
  35. 35.
    Yang, Y., Ma, C., Xu, Y., Wei, Q., Imtiaz, M., Lan, H., et al. (2014). A zinc finger protein regulates flowering time and abiotic stress tolerance in chrysanthemum by modulating gibberellin biosynthesis. The Plant Cell, 26(5), 2038–2054.CrossRefGoogle Scholar
  36. 36.
    Wei, Q., Ma, C., Xu, Y., Wang, T., Chen, Y., Lü, J., et al. (2017). Control of chrysanthemum flowering through integration with an aging pathway. Nature Communications, 8(1), 829.CrossRefGoogle Scholar
  37. 37.
    Murashige, T. S., & Skoog, F. A. (1962). A revised medium for rapid growth and bioassays with tobaco tissue cultures. Physiologia Plantarum, 15(3), 473–497.CrossRefGoogle Scholar
  38. 38.
    Dong, B., Deng, Y., Wang, H., Gao, R., Stephen, G. K., Chen, S., et al. (2017). Gibberellic acid signaling is required to induce flowering of chrysanthemums grown under both short and long days. International Journal of Molecular Sciences, 18(6), 1259.CrossRefGoogle Scholar
  39. 39.
    Guo, A., He, K., Liu, D., Bai, S., Gu, X., Wei, L., et al. (2005). DATF: A database of Arabidopsis transcription factors. Bioinformatics, 21(10), 2568–2569.CrossRefGoogle Scholar
  40. 40.
    Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731–2739.CrossRefGoogle Scholar
  41. 41.
    Zhou, F., Lin, Q., Zhu, L., Ren, Y., Zhou, K., Shabek, N., et al. (2013). D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling. Nature, 504(7480), 406–410.CrossRefGoogle Scholar
  42. 42.
    Song, A., Zhu, X., Chen, F., Gao, H., Jiang, J., & Chen, S. (2014). A chrysanthemum heat shock protein confers tolerance to abiotic stress. International Journal of Molecular Sciences, 15(3), 5063–5078.CrossRefGoogle Scholar
  43. 43.
    Gao, H., Song, A., Zhu, X., Chen, F., Jiang, J., Chen, Y., et al. (2012). The heterologous expression in Arabidopsis of a chrysanthemum Cys2/His2 zinc finger protein gene confers salinity and drought tolerance. Planta, 235(5), 979–993.CrossRefGoogle Scholar
  44. 44.
    Chen, Y., Jiang, J., Song, A., Chen, S., Shan, H., Luo, H., et al. (2013). Ambient temperature enhanced freezing tolerance of Chrysanthemum dichrum CdICE1 Arabidopsis via miR398. BMC Biology, 11(1), 121.CrossRefGoogle Scholar
  45. 45.
    Clough, S. J., & Bent, A. F. (1998). Floral dip: A simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. The Plant Journal, 16(6), 735–743.CrossRefGoogle Scholar
  46. 46.
    Wang, H., Chen, S., Jiang, J., Zhang, F., & Chen, F. (2015). Reference gene selection for cross-species and cross-ploidy level comparisons in Chrysanthemum spp. Scientific Reports, 5, 8094.CrossRefGoogle Scholar
  47. 47.
    Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods, 25(4), 402–408.CrossRefGoogle Scholar
  48. 48.
    Du, H., Huang, M., Zhang, Z., & Cheng, S. (2014). Genome-wide analysis of the AP2/ERF gene family in maize waterlogging stress response. Euphytica, 198, 115–126.CrossRefGoogle Scholar
  49. 49.
    Nakano, T., Suzuki, K., Fujimura, T., & Shinshi, H. (2006). Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology, 140(2), 411–432.CrossRefGoogle Scholar
  50. 50.
    Zhang, H., Yang, Y., Zhang, Z., Chen, J., Wang, X. C., & Huang, R. (2008). Expression of the ethylene response factor gene TSRF1 enhances abscisic acid responses during seedling development in tobacco. Planta, 228(5), 777–787.CrossRefGoogle Scholar
  51. 51.
    Han, Y. C., Kuang, J. F., Chen, J. Y., Liu, X. C., Xiao, Y. Y., Fu, C. C., et al. (2016). Banana transcription factor MaERF11 recruits histone deacetylase MaHDA1 and represses the expression of MaACO1 and expansins during fruit ripening. Plant Physiology, 171(2), 1070–1084.Google Scholar
  52. 52.
    Koyama, T., Nii, H., Mitsuda, N., Ohta, M., Kitajima, S., Ohme-takagi, M., et al. (2013). A regulatory cascade involving class II ethylene response factor transcriptional repressors operates in the progression of leaf senescence. Plant Physiology, 162(2), 991–1005.CrossRefGoogle Scholar
  53. 53.
    Li, Y., Zhu, B., Xu, W., Zhu, H., Chen, A., Xie, Y., et al. (2007). LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato. Plant Cell Reports, 26(11), 1999–2008.CrossRefGoogle Scholar
  54. 54.
    Lee, J. M., Joung, J. G., McQuinn, R., Chung, M. Y., Fei, Z., Tieman, D., et al. (2012). Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation. The Plant Journal, 70(2), 191–204.CrossRefGoogle Scholar
  55. 55.
    Trujillo, L. E., Sotolongo, M., Menendez, C., Ochogavia, M. E., Coll, Y., Hernandez, I., et al. (2008). SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants. Plant Cell Physiology, 49(4), 512–525.CrossRefGoogle Scholar
  56. 56.
    Zhu, X., Qi, L., Liu, X., Cai, S., Xu, H., Huang, R., et al. (2014). The wheat ethylene response factor transcription factor pathogen-induced ERF1 mediates host responses to both the necrotrophic pathogen Rhizoctonia cerealis and freezing stresses. Plant Physiology, 164(3), 1499–1514.CrossRefGoogle Scholar
  57. 57.
    Gu, C., Guo, Z. H., Hao, P. P., Wang, G. M., Jin, Z. M., & Zhang, S. L. (2017). Multiple regulatory roles of AP2/ERF transcription factor in angiosperm. Botanical Studies, 58(1), 6.CrossRefGoogle Scholar
  58. 58.
    Hiratsu, K., Matsui, K., Koyama, T., & Ohme-takagi, M. (2003). Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. The Plant Journal, 34(5), 733–739.CrossRefGoogle Scholar
  59. 59.
    Shim, J. S., & Imaizumi, T. (2015). Circadian clock and photoperiodic response in Arabidopsis: From seasonal flowering to redox homeostasis. Biochemistry, 54(2), 157–170.CrossRefGoogle Scholar
  60. 60.
    Fornara, F., De, M. A., & Coupland, G. (2010). Snapshot: Control of flowering in Arabidopsis. Cell, 141(3), 550–550.e2.CrossRefGoogle Scholar
  61. 61.
    Simpson, G. G., & Dean, C. (2002). Arabidopsis, the rosetta stone of flowering time? Science, 296(5566), 285–289.CrossRefGoogle Scholar
  62. 62.
    Song, Y. H., Ito, S., & Imaizumi, T. (2013). Flowering time regulation: Photoperiod- and temperature-sensing in leaves. Trends in Plant Science, 18(10), 575–583.CrossRefGoogle Scholar
  63. 63.
    Amasino, R. M., & Michaels, S. D. (2010). The timing of flowering. Plant Physiology, 154(2), 516–520.CrossRefGoogle Scholar
  64. 64.
    Choi, K., Kim, J., Hwang, H. J., Kim, S., Park, C., Kim, S. Y., et al. (2011). The frigida complex activates transcription of FLC, a strong flowering repressor in Arabidopsis, by recruiting chromatin modification factors. The Plant Cell, 23(1), 289–303.CrossRefGoogle Scholar
  65. 65.
    Moon, J., Suh, S. S., Lee, H., Choi, K. R., Hong, C. B., Paek, N. C., et al. (2003). The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. The Plant Journal, 35(5), 613–623.CrossRefGoogle Scholar
  66. 66.
    Andrés, F., Porri, A., Torti, S., Mateos, J., Romera-Branchat, M., García-Martínez, J. L., et al. (2014). Short vegetative phase reduces gibberellin biosynthesis at the Arabidopsis shoot apex to regulate the floral transition. Proc Natl Acad Sci USA, 111(26), 2760–2769.CrossRefGoogle Scholar
  67. 67.
    Irish, V. F. (2010). The flowering of Arabidopsis flower development. The Plant Journal, 61(6), 1014–1028.CrossRefGoogle Scholar
  68. 68.
    Lee, J., & Lee, I. (2010). Regulation and function of SOC1, a flowering pathway integrator. Journal of Experimental Botany, 61(9), 2247–2254.CrossRefGoogle Scholar
  69. 69.
    Lee, J., Oh, M., Park, H., & Lee, I. (2008). SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LEAFY. The Plant Journal, 55(5), 832–843.CrossRefGoogle Scholar
  70. 70.
    Mandel, M. A., & Yanofsky, M. F. (1995). A gene triggering flower formation in Arabidopsis. Nature, 377(6549), 522–524.CrossRefGoogle Scholar
  71. 71.
    Kaufmann, K., Wellmer, F., Muiño, J. M., Ferrier, T., Wuest, S. E., Kumar, V., et al. (2010). Orchestration of floral initiation by APETALA1. Science, 328(5974), 85–89.CrossRefGoogle Scholar
  72. 72.
    Wigge, P. A., Kim, M. C., Jaeger, K. E., Busch, W., Schmid, M., Lohmann, J. U., et al. (2005). Integration of spatial and temporal information during floral induction in Arabidopsis. Science, 309(5737), 1056–1059.CrossRefGoogle Scholar
  73. 73.
    Bowman, J. L., Smyth, D. R., & Meyerowitz, E. M. (1989). Genes directing flower development in Arabidopsis. The Plant Cell, 1(1), 37–52.CrossRefGoogle Scholar
  74. 74.
    Shannon, S., & Meeks-wagner, D. R. (1993). Genetic interactions that regulate inflorescence development in Arabidopsis. The Plant Cell, 5(6), 639–655.CrossRefGoogle Scholar
  75. 75.
    Fu, J., Yang, L., & Dai, S. (2015). Identification and characterization of the CONSTANS-like gene family in the short-day plant Chrysanthemum lavandulifolium. Molecular Genetics and Genomics, 290(3), 1039–1054.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Xiaojuan Xing
    • 1
  • Jiafu Jiang
    • 1
  • Yaoyao Huang
    • 1
  • Zixin Zhang
    • 1
  • Aiping Song
    • 1
  • Lian Ding
    • 1
  • Haibing Wang
    • 1
  • Jianjun Yao
    • 2
  • Sumei Chen
    • 1
  • Fadi Chen
    • 1
  • Weimin Fang
    • 1
  1. 1.Key Laboratory of Landscape Agriculture, College of Horticulture, Ministry of AgricultureNanjing Agricultural UniversityNanjingRepublic of China
  2. 2.Shanghai Honghua Horticulture Co. Ltd.ShanghaiChina

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