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Evolution of DNA binding domain of LEAFY: from angiosperms to mosses

Introduction

Figure 1. Schematic presentation LEAFY regulatory roles in controlling floral organ identity (A) and the ABC model in Arabidopsis thaliana (B).

FLORICAULA/LEAFY (FLO/LFY) genes encode a plant specific transcription factor family that controlling floral fate of reproductive phase. ^[1][2]. In the plant model system Arabidopsis thaliana , ‘’LFY’’ also acts upstream of floral homeotic genes to modulate organ identity. ^[3] LFY activates the organ identity genes by binding to promoter regions of floral organ identity genes. LFY can directly bind to the promoter to APELATA1 (AP1), while co-regulators UNUSUAL FLORAL ORGANS (UFO) ^[4] and WUSCHEL (WUS)^[5] are required for increment of binding affinity to promoter regions of APELATA3 (AP3) and AGAMOUS (AG), respectively. The exact mechanism how LFY binds to these promoters has yet to be well elucidated until the first structure report about and ^[6] . Among land plants, FLO/LFY homologs share a highly conserved DNA binding region that a hypothesis claimed substitution in this domain might result in the functional divergence^[7] . Recently, a new study provided new insights of structural basis of LEAFY evolution by changing DNA binding activity^[8].

spinqualitylabelsall models Structure of LFY binding with AP1 and AG promoter region (PDB entry: 2VY1/2VY2) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image Contents 1 Structural Basis of LEAFY binding 1.1 General information about the structure 1.2 Site specific DNA recognition is conducted by a HTH-like motif 1.3 DNA binding required cooperative dimerization Structural Basis of LEAFY binding Figure 2. Site specific recognition of LFY protein at major (A) and minor (B) grooves. Assembly of PDB entry 2VY1 were obtained from PISA server and further visualized by Pymol. Red arrows marked site specific hydrogen bonds. General information about the structure The LFY gene encodes a 424 amino acids protein that containing two domains. The N-terminal domain of LFY has been proved mediating homodimerization (ref) and it is also thought to be responsible for transcriptional activation [9]. The C-terminal consensus is highly conserved among land species and functioning as DNA-binding domain. Two DNA-protein binding structure for LEAFY were first published by Hame et al. 2008 [1][2]. These two structures include a recombinant C-terminal domain of LEAFY expressed by Escherichia coli strain RosettaBlue (DE3) and a short nucleotide structure from AP1 or AG promoter region. Final models of LFY-pAP1 and LFY-pAG were solved at 2.1 Å and 2.3 Å by X-ray diffraction and deposited as PDB entry /. Site specific DNA recognition is conducted by a HTH-like motif The general structure of LEAFY DNA binding domain consists 2 at the beginning followed by 7 . A (HTH) motif can be found between α2 and α3 helices, which is recruited to the of the binding DNA. There are two amino acid at this motif, on α2 and on α3 directly mediate site specific recognition with at the DNA strand. These two recognition sites were further validated by electrophoresis mobility shift assay (EMSA): mutation at either Asn 291 or Lys 307 dramatically decrease binding affinity to pAP1. In the minor groove, site specific recognition is conducted by , which is at the beginning of this structure. Arabidopsis intermediate mutant lfy-4 (P240L) and lfy-5 (T244M) were located near this site and validate the function in planta. The super position of specific recognition sites is summaries at right figure produced by PyMol. DNA binding required cooperative dimerization Figure 3. Three residues mediate homodimerization of LFY dimerization at pAP1 site. Assembly of PDB entry 2VY1 were obtained from PISA server and further visualized by Pymol. Transcription factors tent to form homodimer or heterodimer to increase the binding specificity and affinity. Experimental evidence indicates a potential LFY dimer on the binding site. Crystal structure proved that LFY can form dimers at both and sites. The binding affinity of LFY protein dimer binds increased by 90-fold compared to the first LFY monomer in EMSA assay. Detailed structure revealed that the contact of two dimerized protein is mediated by located at on helix () and one loop () at the other protein. Hydrogen bonds can be formed between and / are essential for this dimeriation. The detailed interaction is shown in the right figure produced by Pymol. Mutation in any of these three amino acids abolished the binding in EMSA assay. Recently, another experiment showing that despite these three residues, the entire N-terminal consensus is critical important for stabilizing the homodimerization, where strong physical interaction can be found by GST-pull down, Y2H and BiFC experiment at in vitro, in vivo and in planta level.

LEAFY Evolution

Reference

1. ↑ ^{1.0 1.1} Detlef Weigel, John Alvarez, David R. Smyth, Martin F. Yanofsky, Elliot M. Meyerowitz, LEAFY controls floral meristem identity in Arabidopsis. Cell 69 :843-859, http://dx.doi.org/10.1016/0092-8674(92)90295-N.
2. ↑ ^{2.0 2.1} Coen, E.S., Romero, J.M., Doyle, S., Elliot, R., Murphy, G. & Carpenter, R. (1990) floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell 63: 1311–1322 http://dx.doi.org/10.1016/0092-8674(90)90426-F
3. ↑ Irish, V. F. (2010), The flowering of Arabidopsis flower development. The Plant Journal, 61: 1014–1028. http://dx.doi.org/10.1111/j.1365-313X.2009.04065.x
4. ↑ Chae, E., Tan, Q.K., Hill, T.A. & Irish, V.F. 2008. An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development. Development 135:1235-45 http://dx.doi.org/10.1242/dev.015842
5. ↑ HONG, R.L., HAMAGUCHI, L., BUSCH, M.A. and WEIGEL, D. (2003). Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing. Plant Cell 15: 1296-1309. http://dx.doi.org/10.1105/tpc.009548
6. ↑ Hames, C., Ptchelkine, D., Grimm, C., Thevenon, E., Moyroud, E., Gérard, F. Martiel, J.L., Benlloch, R., Parcy, F. & Müller, C.W. 2008. Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins. EMBO Journal 27:2628-2637. http://dx.doi.org/10.1038/emboj.2008.184
7. ↑ MAIZEL, A., BUSCH, M.A., TANAHASHI, T., PERKOVIC, J., KATAO, M., HASEBE, M. and WEIGEL, D. (2005). The floral regulator LEAFY evolves by substitutions in the DNA binding domain. Science 308: 260-263. http://dx.doi.org/10.1126/science.1108229
8. ↑ Sayou, C., Monniaux, M., Nanao, M.H., Moyroud, E., Brockington, S.F., Thévenon, E., Chahtane, H., Warthmann, N., Melkonian, M., Zhang, Y., Wong, G., Weigel, D., Parcy, F. and Dumas, R. 2014. A Promiscuous Intermediate Underlies the Evolution of LEAFY DNA Binding Specificity Science 343: 645-648 http://dx.doi.org/10.1126/science.1248229
9. ↑ Cite error: Invalid <ref> tag; no text was provided for refs named Hames_et_al.