Research ArticleArticles

Dimerization in LBD16 and LBD18 Transcription Factors Is Critical for Lateral Root Formation

Han Woo Lee, Na Young Kang, Shashank K. Pandey, Chuloh Cho, Sung Haeng Lee, Jungmook Kim

Published May 2017. DOI: https://doi.org/10.1104/pp.17.00013

Abstract

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (hereafter referred to as LBD) are plant-specific transcription factors that play important roles in a plethora of plant growth and development. The leucine (Leu) zipper-like coiled-coil motif in the lateral organ boundaries domain of the class I LBD proteins has been proposed to mediate protein dimerization, but it has not been experimentally assessed yet. LBD16 and LBD18 have been well characterized to play important roles in lateral root development in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of the coiled-coil motif in the dimerization of LBD16 and LBD18 and in transcriptional regulation and biological function. We built the molecular models of the coiled coil of LBD16 and LBD18, providing the probable Leu zipper models of the helix dimer. Using a variety of molecular techniques, such as bimolecular fluorescence complementation, luciferase complementation imaging, GST pull down, and coimmunoprecipitation assays, we showed that the conserved Leu or valine residues in the coiled-coil motif are critical for the dimerization of LBD16 or LBD18. Using transgenic Arabidopsis plants that overexpress HA:LBD16 or HA:LBD16Q in lbd16 or HA:LBD18 or HA:LBD18Q in lbd18, we demonstrated that the homodimerization of LBD18 mediated by the coiled-coil motif is crucial for transcriptional regulation via promoter binding and for lateral root formation. In addition, we found that the carboxyl-terminal region beyond the coiled-coil motif in LBD18 acts as an additional dimerization domain. These results provide a molecular basis for homodimerization and heterodimerization among the 42 Arabidopsis LBD family members for displaying their biological functions.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (ASLs; hereafter referred to as LBD) are plant-specific transcription factors characterized by a highly conserved unique lateral organ boundaries (LOB) domain in the N terminus and play crucial roles in defining lateral organs (Majer and Hochholdinger, 2011; Xu et al., 2016). LBD proteins are involved in diverse aspects of plant development, including embryo, root, leaf, pollen, photomorphogenesis, secondary growth, plant defense, plant regeneration, and inflorescence development.

In Arabidopsis (Arabidopsis thaliana), LBD16 and LBD18 along with LBD29 and LBD33 have critical and distinct roles in auxin-regulated lateral root development (Okushima et al., 2007; Lee et al., 2009, 2013a, 2013b, 2015; Berckmans et al., 2011; Feng et al., 2012; Goh et al., 2012). LBD16 is involved in initiating nuclear migration and asymmetric division of lateral root founder cells for lateral root initiation (Goh et al., 2012). LBD18 plays a role in lateral root initiation and also in the emergence of lateral roots (Lee et al., 2009, 2013a, 2015; Berckmans et al., 2011; Lee and Kim, 2013). LBD18 mediates lateral root initiation through transcriptional activation of E2Fa (Berckmans et al., 2011). LBD18 activates the expression of other cell cycle genes such as CYCLIN-DEPENDENT KINASE A1;1 and CYCLINB1;1 (Lee et al., 2015). LBD18 up-regulates EXPANSIN14 (EXP14) by directly binding to the EXP14 promoter and indirectly activates EXP17 and other EXP genes for cell wall loosening and POLYGALACTURONASE at a later time point for cell wall remodeling, promoting lateral root emergence (Lee et al., 2013a, 2015).

The Arabidopsis LBD proteins are grouped into two classes according to their structural features (Shuai et al., 2002). The class I proteins comprise 36 members (LOB, LBD1–LBD33, LBD35, and LBD36) and harbor an LOB domain similar to that in the LOB protein, whereas the class II proteins comprise six members (LBD37–LBD42) and are less similar to the class I proteins but share a conserved amino acid sequence with limited conservation outside the LOB domain (Majer and Hochholdinger, 2011; Coudert et al., 2013). The LOB domain contains several conserved motifs, such as a four-Cys motif, a Gly-Ala-Ser block, and a predicted coiled-coil motif with LX6LX3LX6L spacing similar to the Leu zipper (Shuai et al., 2002). The LOB domain is critical for DNA binding and biological function (Husbands et al., 2007; Lee et al., 2013b). The motifs responsible for the nuclear targeting of LBD16 are mapped to two distinct regions comprising an atypical nuclear localization signal in the coiled-coil motif and a monopartite-like nuclear localization signal in the C-terminal region (Kim and Kim, 2012). The C-terminal region from LBD16, LBD18, or LBD30 activates reporter gene expression when fused to the Gal4 DNA-binding domain in yeast and in Arabidopsis protoplasts (Borghi et al., 2007; Lee et al., 2013a). LBD18 activates the expression of EXP14 by directly binding to the EXP14 promoter (Lee et al., 2013a). G-BOX-BINDING FACTOR INTERACTING PROTEIN1, a transcriptional coactivator, enhances the transcriptional activity of LBD18 (Lee et al., 2014). Thus, some class I LBD proteins act as DNA-binding transcriptional activators. In contrast, LBD6/AS2 acts as a repressor for KNOX genes (Guo et al., 2008).

The protein dimerization of transcription factors is critical for the modulation of DNA-binding affinity and specificity, contributing to transcriptional regulation (Funnell and Crossley, 2012). Protein dimerization was observed among several class I LBD proteins, including homodimerization of LBD16 and LBD18, heterodimerization of LBD10-LBD27, LBD18-LBD33, and LBD30-LBD6 dimers, and combinatorial interactions of LBD10, LBD22, LBD25, LBD27, and LBD36 (Berckmans et al., 2011; Majer et al., 2012; Rast and Simon, 2012; Lee et al., 2013b; Kim et al., 2015, 2016). Although the predicted Leu zipper-like coiled-coil motif in the LOB domain of the class I LBD proteins has been proposed to mediate protein dimerization among LBD proteins (Shuai et al., 2002), it has not been experimentally assessed yet. Moreover, the class II LBD proteins and some class I LBD proteins such as LBD2, LBD26, and LBD34 have no predicted coiled coil. In this work, we demonstrate that the coiled-coil motif is crucial for the homodimerization of LBD16 and LBD18 required for transcriptional regulation in promoting lateral root formation. In addition, the C-terminal region beyond the coiled-coil motif was shown to act an additional dimerization domain in LBD18. This result provides a molecular basis for homodimerization and heterodimerization among the 42 members of the Arabidopsis LBD family for displaying their biological functions.

RESULTS

Modeling of the Zipper-Like Conformation of LBD16 and LBD18

To understand the role of the coiled-coil motif in the dimerization of LBD proteins and their transcriptional regulation and biological function, we focused on LBD16 and LBD18, which have been well characterized molecularly and biologically to play important roles in lateral root development in Arabidopsis. We first built the molecular models for the coiled coil of LBD16 and LBD18 using the Phyre2 suit (Kelley and Sternberg, 2009) with known protein structures. The amino acid sequences 91 to 120 for LBD16 and 111 to 150 for LBD18 were provided for Phyre2 to generate the most probable molecular models. The helix-loop-helix Leu zipper in the STEROL REGULATORY ELEMENT-BINDING PROTEIN2 (SREBP2) showed the highest homology to the coiled-coil motif of LBD18 and LBD16, with confidence of 74.7% and 63.1%, respectively. The predicted helix model was then aligned to chains C and D (sequence 374–402) in the cocrystal structure of importin-β with SREBP2 (Protein Data Bank code 1UKL), which provides the probable Leu zipper model of the helix dimer (Fig. 1A). Further optimization to minimize the side chain repulsion between two helices was performed. Each helix of the model from LBD18 has 7.5 turns, and the two helices form a zipper-like dimer connected by the extended hydrophobic interactions. Amino acid residues Leu-120, Leu-127, Leu-134, Leu-138, and Leu-141 from each helix participate mainly in the formation of the Leu zipper-like conformation. To compensate for the lack of authenticity of the domain formation, three additional hydrophobic residues, Ile-117, Val-124, and Val-131, fill the gaps between the Leu molecules. On the other hand, the LBD16 model shows non-Leu residues in dominant zipper formation (Fig. 1B). The LBD16 helix is one turn shorter than the LBD18 helix and contains only two Leu residues (residues 97 and 105) in the helix. Four additional hydrophobic amino acid residues (Ile-95, Ile-116, Val-102, and Val-109) create the hydrophobic interface between the helices.

Figure 1.
Figure 1.

Structure prediction of the zipper-like conformation of the LBD16 and LBD18 dimer using the Phyre2 server. A, LBD18. a and b indicate the helices predicted from amino acids 111 to 150 of each monomer. B, LBD16. a and b indicate the helices predicted from amino acids 91 to 120 of each monomer.

Critical Role of the Coiled-Coil Motif in the Dimerization of LBD16 or LBD18

LBD16 and LBD18 contain the L98X6LX3VX6I116 and L120X6LX3VX6L138 motifs, respectively, where the superscript and subscript indicate the amino acid number and the number of amino acids in the spacing, respectively, and X indicates the presence of any amino acid. To determine the role of the predicted coiled-coil motif for the dimerization of LBD16 or LBD18, we changed the conserved Leu, Val, or Ile residues of the coiled-coil motif into the Pro residue by site-directed mutagenesis and investigated the effects of these mutations on the protein-protein interactions using the bimolecular fluorescence complementation (BiFC) assay (Walter et al., 2004). The effects of single or double mutations on the protein-protein interactions of LBD16 or LBD18 varied depending upon whether the position of the amino acid residue changed. However, any combination of triple mutations significantly reduced the protein-protein interactions (Supplemental Figs. S1–S4). Quadruple mutations greatly reduced the protein interactions of LBD16Q or LBD18Q (Fig. 2). We next used the firefly luciferase complementation imaging (LCI) assay with Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana (Chen et al., 2008) for quantitative detection of the protein-protein interactions between LBD18Q and LBD18Q or between LBD18Q and LBD18 compared with those of wild-type LBD18 in vivo. Leaves coexpressing different constructs consisting of LBD18 or LBD18Q fused with the N-terminal luciferase (NLuc) and LBD18 or LBD18Q fused with the C-terminal luciferase (CLuc) were examined for LUC activity. The LUC images captured by CCD camera and quantification of the LUC activities showed that the relative luminescence intensity caused by the protein-protein interactions between LBD18Q and LBD18 or between LBD18Q and LBD18Q decreased to 22% or 40% of the levels caused by interaction with wild-type LBD18, whereas all negative controls exhibited undetectable luminescence (Fig. 3, A and B). The GST pull-down assays showed that the pull down of 35S-labeled LBD18Q by the GST:LBD18 recombinant protein (Fig. 3C, lane 6) was much weaker than that of 35S-labeled LBD18 (Fig. 3C, lane 5). Moreover, coimmunoprecipitation assays using Arabidopsis mesophyll protoplasts showed that quadruple mutations in the coiled-coil motif of LBD18 greatly diminished immunoprecipitates of HA:LBD18Q with MYC:LBD18 (Fig. 3D). Taken together, these results demonstrated that the conserved Leu or Val residues of the coiled-coil motif are critical for the dimerization of LBD16 or LBD18.

Figure 2.
Figure 2.

BiFC analysis of the effects of quadruple mutations in the coiled-coil motif of LBD16 or LBD18 on protein-protein interactions with Arabidopsis protoplasts. A, Conserved motif structures of LBD16 and LBD18. Black lines indicate the locations of amino acids where the conserved Leu, Val, or Ile residues were changed into Pro residues by site-directed mutagenesis. C block, Four-Cys motif with CX2CX6CX3C spacing; GAS block, Gly-Ala-Ser block; CC, predicted coiled-coil motif with LX6LX3LX6L spacing; AD, transactivation domain. The numbers indicate amino acid numbers. Red lines in the coiled-coil motif indicate quadruple mutations of the conserved Leu, Val, or Ile residues into Pro residues. LBD16Q and LBD18Q indicate LBD16L98P,L105P,V109P,I116P and LBD18L120P,L127P,V131P,L138P, respectively. B, Epifluorescence (YFP), autofluorescence (chloroplast), and merged images. Bars = 10 μm.

Figure 3.
Figure 3.

Analysis of the protein-protein interactions between LBD18 and LBD18Q. A, LCI assays in N. benthamiana. The top shows the conserved motif structures of LBD18 and the locations of the mutations in LBD18Q. The bottom shows LUC images of N. benthamiana leaves coinfiltrated with A. tumefaciens harboring Pro35S:CLuc, Pro35S:CLuc, Pro35S:NLuc:LBD18, and/or Pro35S:CLuc:LBD18Q. The pseudocolor bar shows the range of luminescence intensity from weak (blue) to strong (red). B, Quantification of the LCI assays shown in A. Data are means ± se determined from three independent biological replicates. Asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**). C, GST pull-down assays. GST:LBD18 recombinant proteins were incubated with [35S]LBD18 or [35S]LBD18Q for pull-down assays. GST proteins were used as a control. D, Coimmunoprecipitation assays. Protoplasts isolated from 2-week-old Arabidopsis plants were transfected with YFPN:LBD18 and YFPC:LBD18 (lane 1) or with YFPN:LBD18 and YFPC:LBD18Q (lane 2) plasmid DNA. Samples prepared after incubation were precipitated with anti-c-Myc agarose, and immunoblot analysis was performed with monoclonal anti-HA or anti-c-Myc antibody.

Homodimerization of LBD16 or LBD18 Is Required for the Function in Lateral Root Formation

To investigate the biological role of the coiled-coil motif-mediated dimerization of LBD16 or LBD18, we constructed transgenic Arabidopsis that overexpresses HA:LBD16 or HA:LBD16Q in the lbd16 mutant (Pro35S:HA:LBD16:GR/lbd16 or Pro35S:HA:LBD16Q:GR/lbd16) or HA:LBD18 or HA:LBD18Q in the lbd18 mutant (Pro35S:HA:LBD18:GR/lbd18 or Pro35S:HA:LBD18Q:GR/lbd18), respectively, under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter in frame with the hormone-binding domain of the glucocorticoid receptor (GR) and examined lateral root formation. We selected three different transgenic lines expressing similar levels of HA:LBD16Q or HA:LBD18Q compared with wild-type HA:LBD16 or HA:LBD18 by immunoblot analysis (Supplemental Fig. S5). Dexamethasone (DEX) treatment of Pro35S:HA:LBD16:GR/lbd16 or Pro35S:HA:LBD18:GR/lbd18 plants rescued the lateral root density of lbd16 or lbd18 to wild-type levels, whereas DEX treatment of Pro35S:HA:LBD16Q:GR/lbd16 or Pro35S:HA:LBD18Q:GR/lbd18 plants did not (Fig. 4, A and B). As EXP14 has been identified previously as a direct target of LBD18 (Lee et al., 2013a), we determined the expression levels of EXP14 with or without DEX and found that quadruple mutations in the coiled-coil motif of LBD18 abolished or greatly reduced EXP14 expression in two different transgenic lines by DEX compared with that of wild-type LBD18 (Fig. 4C). Marginal but significant levels of EXP14 expression noted in line 14-1 of Pro35S:HA:LBD18Q:GR/lbd18 transgenic plants (Fig. 4C) indicated that certain threshold expression levels of target genes regulated by LBD18 are required for LBD18 to mediate its function in lateral root formation. To examine if the quadruple mutations in the coiled-coil motif of LBD18 affect transcription-activating function, transient gene expression assays with Arabidopsis protoplasts were performed with a reporter plasmid harboring the LUC reporter gene fused with the Gal4(3X) promoter element and an effect plasmid harboring LBD18 or LBD18Q fused with the Gal4 DNA-binding domain (LBD18:GD or LBD18Q:GD) under the control of the CaMV 35S promoter (Fig. 5). However, both LBD18:GD and LBD18Q:GD displayed similar levels of the transactivation capability of the LUC reporter gene, indicating that the quadruple mutations did not alter the transactivation capability of LBD18. Taken together, these results demonstrated that homodimerization of LBD18 mediated by the coiled-coil motif is crucial for transcriptional regulation via proper promoter binding for displaying biological function.

Figure 4.
Figure 4.

Analysis of the role of the dimerization of LBD16 or LBD18 mediated by the coiled-coil motif in lateral root formation in Arabidopsis. A and B, Complementation of lbd16 by LBD16:GR or LBD16Q:GR (A) and lbd18 by LBD18:GR or LBD18Q:GR (B). Numbers above the lines indicate the line numbers of transgenic plants. Plants were grown vertically for 7 d after germination in the absence or presence of 10 µm DEX, and lateral root numbers per unit of root length (cm; lateral root density) measured were plotted. Data are means ± sd determined from three independent biological replicates. Asterisks denote statistical significance at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). C, Expression analysis of EXP14 in Pro35S:LBD18Q:GR/lbd18 and Pro35S:LBD18:GR/lbd18 plants. Seven-day-old seedlings were treated with or without 10 µm DEX for 4 h, and then roots were harvested for real-time reverse transcription (RT)-PCR analysis. Data are means ± se determined from four independent biological replicates (each biological replicate was estimated as the average of two technical RT-PCR replicates). Asterisks denote statistical significance at P < 0.001 (***). Numbers above the lines indicate the line numbers of transgenic plants.

Figure 5.
Figure 5.

Transcriptional activities of LBD18 and LBD18Q via the EXP14 promoter in Arabidopsis mesophyll protoplasts. A, Schematic diagrams showing reporter plasmids and effector plasmids for transient gene expression assays. The effector plasmid consisted of the Gal4 DNA-binding domain (GD) fused to the DNA sequences encoding the full-length of LBD18 or LBD18L120P,L127P,V131P,L138P under the control of the CaMV 35S promoter (Pro35S:Ω:LBD18 or Pro35S:Ω:LBD18L120P,L127P,V131P,L138P). The reporter construct consisted of three copies of the Gal4 DNA-binding domain elements fused to the LUC reporter gene [ProGal4(3X):LUC]. The reporter construct consisting of LUC driven by the CaMV 35S promoter (Pro35S:GUS) was used for a transfection control. B, Transient gene expression assays. All LUC activities are presented after normalizing to GUS activity. Data are means ± se determined from three independent biological replicates. Asterisks denote statistical significance at P < 0.01 (*) and P < 0.001 (**).

The C-Terminal Region beyond the Coiled-Coil Motif Functions as an Important Dimerization Domain of LBD18

Forty percent of the LBD18Q proteins could still dimerize with each other compared with the levels of homodimerization of the LBD18 proteins, and LBD18Q also heterodimerized with LBD18 to some extent (Figs. 2 and 3B). Although it is possible that quadruple mutations in the coiled-coil motif may not be sufficient to completely block protein dimerization, an additional domain other than the coiled-coil motif may be required for the formation of fully stable LBD18 dimer. To identify an additional protein interaction domain in LBD18, we conducted quantitative LCI assays with various fragments of LBD18 (Fig. 6). Several LBD18 fragments were made based on the conserved motifs and functional domains and fused with the N-terminal fragment or C-terminal fragment of luciferase (NLuc or CLuc; Fig. 6A). As expected, any of the LBD18 polypeptide harboring the coiled-coil motif homodimerizes (Fig. 6). Interestingly, we found that the C-terminal fragment of LBD18 (construct 4) formed a homodimer stronger than the full-length LBD18 or the other LBD18 fragments. When we examined the heterodimerization of various LBD18 fragments with the full-length LBD18, the C-terminal domain (construct 4) gave the highest luminescence intensity among the LBD18 fragments tested (Fig. 6, D and E). The N-terminal region without both the coiled-coil motif and the C-terminal domain completely lost protein dimerization capability (Fig. 6, C and E). Taken together, these results indicate that the C-terminal domain beyond the coiled-coil motif acts as an additional dimerization domain in LBD18 and that both the coiled-coil motif and the C-terminal domain are required for the formation of fully active LBD18 dimer.

Figure 6.
Figure 6.

Analysis of the protein interaction domain of LBD18 using LCI assays. A, Diagram showing the polypeptide fragments of LBD18 used for LCI assays. The numbers on the LBD18 diagram and in parentheses indicate the positions of amino acids in the polypeptide and the fragments, respectively. B, LUC images for the homodimerization of various LBD18 polypeptide fragments. The numbers at top indicate the LBD18 polypeptide fragments as shown in Figure 1H. NLuc, N-terminal luciferase; CLuc, C-terminal luciferase. C, Quantification of the LCI assays in B. Data are means ± se determined from three independent biological replicates. D, LUC images for the protein-protein interactions between LBD18 and various LBD18 polypeptide fragments. E, Quantification of the LCI assays in D. Data are means ± se determined from three independent biological replicates. Asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**).

DISCUSSION

Several classes of transcription factors, such as some basic-region Leu zipper (bZIP) and basic helix-loop-helix proteins, can only function as dimers (White, 2001). Dimerization was observed among the LBD family members (Berckmans et al., 2011; Majer et al., 2012; Rast and Simon, 2012; Lee et al., 2013a, 2013b; Kim et al., 2015, 2016). While the coiled-coil motif has been proposed to mediate protein-protein interactions within the class I LBD proteins, there is no report on the role of the coiled-coil motif in LBD protein dimerization. Moreover, whether dimer formation of LBD proteins is necessary for exhibiting their biological functions is not known. In this study, we demonstrated that the conserved Leu or Val residues in the coiled-coil motif of LBD16 and LBD18 are critical for dimer formation and that the homodimerization of LBD18 is crucial for its transcriptional and biological function in controlling lateral root formation in Arabidopsis.

The Leu zipper is the dimerization domain of the bZIP class of eukaryotic transcription factors and a left-handed parallel coiled coil (Landschulz et al., 1988; Glover and Harrison, 1995). Each monomer in the Leu zipper has a structural repeat of two α-helical turns termed heptad (Llorca et al., 2014). In each heptad, seven amino acids designated with letters from a to g positions are arranged around the two helices. Amino acids in the a and d positions lie on the same side of the α-helix and are typically hydrophobic. These hydrophobic amino acids interact with hydrophobic amino acids in the same positions of the second α-helix of the Leu zipper. Typically, the d position contains Leu, which is the most stabilizing factor for the dimerization (Moitra et al., 1997). According to the letter designation of the Leu zipper of bZIP, LBD18 has four Leu amino acids at position d and hydrophobic amino acids, Ile, Val, Val, and Leu, at position a in four heptads, whereas LBD16 has Leu, Leu, Met, and His at position d and hydrophobic amino acids, Ile, Val, Val, and Ile, at position a in four heptads, indicating that the coiled coil of LBD16 is not a typical Leu zipper, whereas the coiled coil of LBD18 is similar to the Leu zipper (Supplemental Fig. S6). Our structure prediction using Phyre2 indicated that the two coiled-coil motifs from each monomer form a Leu zipper-like conformation in both LBD16 and LBD18 (Fig. 1). Consistent with this, the homodimerization of LBD16 or LBD18 in BiFC assays was progressively weakened with the increasing number of mutations in Leu or other hydrophobic amino acid residues in the coiled-coil motif (Fig. 2; Supplemental Figs. S1–S4). Using other molecular methods, such as LCI, GST pull down, and coimmunoprecipitation assays, to determine protein-protein interactions, we demonstrated that the conserved Leu or Val residues of the coiled-coil motif are critical for the dimerization of LBD18 (Fig. 3). We observed that the relative luminescence intensity between LBD18Q and LBD18Q is higher than that of LBD18Q and LBD18 (40% to 22%; Fig. 3B). Although it is difficult to justify the biochemical reason for the higher relative luminescence intensity between LBD18Q and LBD18Q than between LBD18Q and LBD18 without x-ray crystal structures of LBD18 and LBD18Q, it may be because two strands of four Pro residues in LBD18Q homodimer may be involved in interacting with each other.

We found that the LBD16 or LBD18 protein harboring quadruple mutations in the coiled-coil motif could not complement the reduced number of lateral root densities of lbd16 or lbd18 mutants even under the control of the CaMV 35S promoter, while wild-type LBD16 or LBD18 could fully rescue the defect in lateral root densities of lbd16 or lbd18 (Fig. 4, A and B). Moreover, these mutations completely or greatly abolished the expression of EXP14, a direct target gene of LBD18, but did not affect the transcription-activating activity of LBD18 in transient gene expression assays with Arabidopsis protoplasts (Figs. 4C and 5). Quadruple mutations in Leu or other hydrophobic amino acids in the coiled-coil motif resulted in a great reduction in protein dimerization activity of both LBD16 and LBD18 but did not completely abolish the dimerization capability of LBD16 and LBD18 (Figs. 2 and 3). The reason why LBD16Q or LBD18Q with residual dimerization capability did not cause any partial rescue in the lbd18 phenotypes may be because the dimer of LBD16Q or LBD18Q may not be in a proper conformation for binding properly to the promoters of target genes and/or because certain threshold expression levels of target genes regulated by LBD18 are required for LBD18 to fulfill its function in lateral root formation. These results together suggest that precise homodimer formation of LBD16 or LBD18 is crucial for binding to the promoters of the target genes and for their biological function. Consistent with this suggestion, we noted previously that a significant dominant mutation effect on lateral root formation was not caused by overexpression of LBD16P87L or LBD18P109L, which lost the DNA-binding activity but could homodimerize and heterodimerize, in lbd16 or lbd18 mutants, indicating that LBD16 and LBD18 mainly function as homodimers to regulate lateral root formation in Arabidopsis (Lee et al., 2013b). While the homodimer formation of LBD16 and LBD18 is critical for lateral root formation, the biological roles of LBD16-LBD18 and LBD18-LBD33 heterodimers (Berckmans et al., 2011; Lee et al., 2013b) in lateral organ development remain to be determined.

Protein-protein interaction studies have shown that some LBD proteins can form homodimers and heterodimers with specificity, whereas other LBD proteins can form exclusively heterodimers (Berckmans et al., 2011; Majer et al., 2012; Rast and Simon, 2012; Lee et al., 2013b; Kim et al., 2015, 2016). LBD22, LBD27, and LBD36 heterodimerize to be localized to the nucleus (Kim et al., 2015, 2016). Heterodimerization between two different LBD proteins can contribute to transcriptional regulation by modulating DNA-binding specificity and the recruitment of their binding partners. Thus, combinatorial association among the LBD family members determined by the unique amino acid sequences in the coiled-coil motifs could be critical for displaying their biological functions.

In addition to the coiled-coil motif for mediating protein-protein interactions between LBD16 or LBD18, we further identified the C-terminal domain beyond the coiled-coil motif as an additional dimerization domain in LBD18 (Fig. 6). This C-terminal region encompassing amino acids 143 to 262 was reported previously to have a transcription-activating function in both yeast and Arabidopsis protoplasts using transient gene expression assays (Lee et al., 2013a). This C-terminal region of LBD18 is rich in Gln and Pro, which are characteristics of the transcriptional activation domain of many transcription factors. As activation domains generally serve as protein-protein interaction domains for coactivators and some components of the basal transcriptional complex (Latchman, 2008), such molecular characteristics of the C-terminal region of LBD18 may render this region as a dimerization domain.

Among the class I LBD proteins, 33 out of 36 proteins were predicted to form a coiled-coil motif with over 90% probability, but LBD2, LBD26, and LBD34 were not predicted to form coiled coils (Shuai et al., 2002). None of the class II proteins were predicted to form coiled-coil structures. We noted that most of the class I LBD proteins are rich in Gln and Pro and that the class II LBD proteins are rich in Pro in their C-terminal regions (Kim and Kim, 2012). It is possible that the C-terminal region of the LBD proteins lacking coiled coils may mediate protein-protein interactions between these LBD proteins as well as between the class I and class II proteins.

MATERIALS AND METHODS

Plant Growth and Tissue Treatment

Arabidopsis (Arabidopsis thaliana) Columbia-0 seedlings were grown and treated as described previously (Park et al., 2002). Surface-sterilized seeds were incubated in the dark at 4°C for 72 h for stratification and were subsequently transferred to a 16-h-light/8-h-dark cycle at 23°C. For quantitative reverse transcription (qRT)-PCR using root tissues, seedlings were grown vertically on 0.5× Murashige and Skoog medium, salts, and vitamins, 1.5% (w/v) Suc, 2.5 mm MES (pH 5.7), and 0.8% phytoagar at 23°C. Seven-day-old seedlings were then treated with mock and DEX for 4 h with gentle shaking in the light at 23°C.

Plasmid Construction and Arabidopsis Transformation

To generate Pro35S:HA:LBD16:GR/lbd16, Pro35S:HA:LBD18:GR/lbd18, Pro35S:HA:LBD16Q:GR/lbd16 and Pro35S:HA:LBD18Q:GR/lbd18 plants, HA:LBD16:GR and HA:LBD18:GR DNA fragments were PCR-amplified from the existing Pro35S:EGFP:LBD16:GR and Pro35S:EGFP:LBD18:GR plasmids (Lee et al., 2013a) using Pfu DNA polymerase (Solgent Daejon, Korea) and subcloned into pGEM T-easy (Promega) vector. NotI and AscI sites were used to generate pENTR-HA:LBD16:GR and pENTR-HA:LBD18:GR plasmids. These plasmids were used to generate quadruple mutations by site-directed mutagenesis in the coiled-coil motif, yielding pENTR-HA:LBD16Q and pENTR-HA:LBD18Q vectors. Resulting pENTR vectors were then used for Gateway LR recombination reaction to clone HA:LBD16:GR, HA:LBD18:GR, HA:LBD16Q:GR and HA:LBD18Q:GR from pENTRTM/SD/D-TOPO to pB7WG2 destination vector, yielding the Pro35S:HA:LBD16:GR, Pro35S:HA:LBD18:GR, Pro35S:HA:LBD16Q:GR and Pro35S:HA:LBD18Q:GR constructs. These vectors were introduced into lbd16 and lbd18 Arabidopsis mutants (Lee et al., 2009) by Agrobacterium-mediated transformation to generate Pro35S:HA:LBD16:GR/lbd16, Pro35S:HA:LBD18:GR/lbd18, Pro35S:HA:LBD16Q:GR/lbd16 and Pro35S:HA:LBD18Q:GR/lbd18 transgenic plants. T3 homozygous transformants were generated and amplified. All constructs were confirmed via DNA sequencing prior to plant transformation. The oligonucleotides used in this study are shown in Supplemental Table S1.

RNA Isolation and qRT-PCR Analysis

Root tissues detached from Arabidopsis plants after treatment with mock and DEX (10 μm) for 4 h were frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated from frozen tissues using the RNeasy Plant Mini Kit (Qiagen) and real-time RT-PCR was conducted with the QuantiTect SYBR Green RT-PCR Kit (Qiagen) in the CFX96 Real-Time System (Bio-Rad) as described previously (Jeon et al., 2010). All real-time RT-PCR assays were conducted in duplicate for the same RNA isolated from each biological experiment. qRT-PCR analysis was carried out for three different biological experiments and subjected to statistical analysis. Statistics were performed with SPSS21. Primer sequences used for RT-PCR and qRT-PCR are shown in Supplemental Table S1.

Western-Blot Analysis

Fifty micrograms of total protein was extracted by the standard procedures (Gusmaroli et al., 2004; Jeon and Kim, 2011), separated by 12% SDS-PAGE, and transferred to immunoblot polyvinylidene difluoride membranes (Bio-Rad). The polyvinylidene difluoride membranes were then subjected to blocking for 2 h, followed by primary antibody binding overnight and secondary antibody binding for 2 h. The blots were detected with ECL substrate in conjunction with the western-blotting detection system (GE Healthcare). The G-Box iChemiXL Gel Documentation System (Syngene) was used to capture fluorescence images of the immunoblots. Monoclonal anti-HA antibody produced in mice was used as a primary antibody at a 1:1,000 dilution, and goat anti-mouse IgG-HRP was employed as a secondary antibody at a dilution of 1:5,000.

BiFC Assays

The yellow fluorescence vectors (YFPN tagged with c-MYC epitope and YFPC tagged with HA epitope) are from Walter et al. (2004). The YFPN:LBD16, YFPN:LBD18, YFPC:LBD16, and YFPC:LBD18 plasmids were described previously (Lee et al., 2013b). The YFPN:LBD16L98P, YFPN:LBD16L105P, YFPN:LBD16V109P, YFPN:LBD16I116P, YFPC:LBD16L98P, YFPC:LBD16L105P, YFPC:LBD16V109P, YFPC:LBD16I116P, YFPN:LBD18L120P, YFPN:LBD18L127P, YFPN:LBD18V131P, YFPN:LBD18L138P, YFPC:LBD18L120P, YFPC:LBD18L127P, YFPC:LBD18V131P, YFPC:LBD18L138P, YFPN:LBD16L98P,L105P, YFPN:LBD16L105P,V109P, YFPN:LBD16V109P,I116P, YFPC:LBD16L98P,L105P, YFPC:LBD16L105P,V109P, YFPC:LBD16V109P,I116P, YFPN:LBD18L120P,L127P, YFPN:LBD18L127P,V131P YFPN:LBD18V131P,L138P, YFPC:LBD18L120P,L127P, YFPC:LBD18L127P,V131P, and YFPC:LBD18V131P,L138P plasmids were generated from the YFPN:LBD16, YFPN:LBD18, YFPC:LBD16, and YFPC:LBD18 plasmids, respectively, by site-directed mutagenesis. The YFPN:LBD16L98P,I116P, YFPC:LBD16L98P,I116P, YFPN:LBD18L120P,L138P, and YFPC:LBD18L120P,V131P plasmids were constructed from the YFPN:LBD16L98P, YFPN:LBD18 L120P, YFPC:LBD16 L98P, and YFPC:LBD18 L120P plasmids by site-directed mutagenesis. The YFPN:LBD16L98P,L105P,V109P, YFPN:LBD16L98P,L105P,I116P, YFPN:LBD16L98P,L109P,I116P, YFPN:LBD16L105P,V109P,I116P, YFPC:LBD16L98P,L105P,V109P, YFPC:LBD16L98P,L105P,I116P, YFPC:LBD16L98P,L109P,I116P, YFPC:LBD16L105P,V109P,I116P, YFPN:LBD18L120P,L127P,V131P, YFPN:LBD18L120P,L127P,L138P, YFPN:LBD18L120P,V131P,L138P, YFPN:LBD18L127P,V131P,L138P, YFPC:LBD18L120P,L127P,V131P, YFPC:LBD18L120P,L127P,L138P, YFPC:LBD18L120P,V131P,L138P, and YFPC:LBD18L127P,V131P,L138P plasmids were generated from the YFPN:LBD16L98P,L105P, YFPN:LBD16L105P,V109P, YFPN:LBD16V109P,I116P, YFPC:LBD16L98P,L105P, YFPC:LBD16L105P,V109P, YFPC:LBD16V109P,I116P, YFPN:LBD18L120P,L127P, YFPN:LBD18L127P,V131P YFPN:LBD18V131P,L138P, YFPC:LBD18L120P,L127P, YFPC:LBD18L127P,V131P, and YFPC:LBD18V131P,L138P plasmids, respectively, by site-directed mutagenesis. The YFPN:LBD16L98P,L105P,V109P,I116P, YFPN:LBD18L120P,L127P,V131P,L138P, YFPC:LBD16L98P,L105P,V109P,I116P, and YFPC:LBD18L120P,L127P,V131P,L138P plasmids were generated from the YFPN:LBD16L98P,I116P, YFPC:LBD16L98P,I116P, YFPN:LBD18L120P,L138P, and YFPC:LBD18L120P,V131P plasmids, respectively, by site-directed mutagenesis. All constructs were verified by DNA sequencing. Site-directed mutagenesis was performed using the proofreading Pfu DNA polymerase (Solgent) under the following PCR conditions: 95ºC for 30 sec for predenaturation, 95ºC for 30 sec for denaturation, 58ºC for LBD16 or 60ºC for LBD18 for 45 sec for annealing, and 72ºC for 13 min for extension, 12 cycles. The PCR products were then digested with DpnI for 3 h at 37ºC, and purified using a Qiagen QIAquick PCR Purification kit prior to E.coil transformation. These plasmids were purified using a Qiagen Plasmid Midi kit prior to protoplast transformation. Protoplasts from Arabidopsis mesophyll cells were prepared as described previously (Lee et al., 2008). YFP fluorescence was monitored by capturing YFP images with a TCS SP5 AOBS spectral confocal and multiphoton microscope system (Leica Microsystems). Confocal images of the YFP fusion proteins were acquired at the Korea Basic Science Institute, Gwangju branch, Korea.

A. tumefaciens-Mediated Transient Expression in Nicotiana benthamiana

To construct vectors for A. tumefaciens-mediated transient expression in N. benthamiana, LBD18 and LBD18L120P,L127P,V131P,L138P DNA was subcloned into pDONRTM201 (Invitrogen) by Gateway BP recombination reaction, yielding pDONR-LBD18 and pDONR-LBD18L120P,L127P,V131P,L138P, respectively. These constructs were subcloned into the pCAMBIA1300-NLuc and pCAMBIA1300-CLuc vectors (Chen et al., 2008) by Gateway LR recombination reaction, yielding NLuc:LBD18, NLuc:LBD18L120P,L127P,V131P,L138P, CLuc:LBD18, and CLuc:LBD18L120P,L127P,V131P,L138P. Various truncated versions of LBD18 in frame with the N terminus of NLuc or the C terminus of CLuc were generated as follows: amino acids 68 to 262 (construct 2 in Supplemental Fig. S8), amino acids 116 to 262 (construct 3), amino acids 144 to 262 (construct 4), amino acids 1 to 143 (construct 5), and amino acids 1 to 115 (construct 6). The PCR-amplified DNA sequences were verified by DNA sequencing. The PCR conditions and primer sequences are shown in Supplemental Table S1. The constructs were mobilized into A. tumefaciens (strain GV3101). A. tumefaciens harboring each vector at OD600 = 0.7 was coinfiltrated into the abaxial epidermal cells of N. benthamiana leaves using a needleless syringe. The plants were incubated for 2 to 3 d at 23°C with 16 h of light/8 h of dark and then subjected to LCI assays.

LCI Assays

The LCI assays were performed as described previously (Chen et al., 2008). One millimolar luciferin (Promega) was added to spray on the leaves of N. benthamiana and then kept in the dark for 5 min to quench the fluorescence. LUC images were captured using a CCD imaging apparatus (cooled high-resolution camera, 16 bits)-based G:BOX Ichemi XL1.4 Imaging System (Syngene). An exposure time of 15 min was used for all images taken. Relative LUC activity was equivalent to the luminescence intensity/leaf area. Three independent experiments were performed for each assay.

GST Pull-Down Assays

The GST and GST:LBD18 plasmids were transformed into bacterial strain BL21-CodonPlus(DE3)-RIL competent cells (Stratagene) and purified as described previously (Lee et al., 2013a). The LBD18 and LBD18L120P,L127P,V131P,L138P cDNAs were amplified by RT-PCR and subcloned into the pCITE-4a vector (Novagen) at the NcoI (N terminus) and XhoI (C terminus) sites by translational fusion. The LBD18 and LBD18L120P,L127P,V131P,L138P polypeptides were labeled with [35S]Met using the TNT-coupled reticulocyte lysate system (Promega). Ten microliters of the 35S-labeled proteins was incubated at 4°C for 3 h with 5 μg of GST or GST:LBD18 proteins already bound to the glutathione-Sepharose 4B beads (GE Healthcare). The beads were washed five times with 1 mL of ice-cold GST lysis buffer (20 mm Tris-Cl, pH 8, 200 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, and 1× Complete protease inhibitor cocktail [2 μg μL−1 aprotinin, 1 μg μL−1 leupeptin, 0.7 μg mL−1 pepstatin, and 25 μg mL−1 phenylmethylsulfonyl fluoride]). The bound proteins were eluted with 40 μL of 1× SDS-PAGE loading buffer by boiling for 5 min at 100°C and then subjected to SDS-PAGE and autoradiography.

Coimmunoprecipitation Assays

Arabidopsis protoplasts isolated from 2-week-old plants were transfected with YFPN:LBD18 and YFPC:LBD18 or with YFPN:LBD18 and YFPC:LBD18Q plasmid DNA as described previously (Lee et al., 2008). Protoplasts (4 × 104 per sample) were incubated for 16 h in the dark at 23°C. Cells were harvested and resuspended in 100 μL of immunoprecipitation buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Nonidet P-40, 50 μm MG132, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors (Kim et al., 2008). Following incubation on ice for 20 min, the extracts were centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was incubated with 40 μL of anti-c-Myc agarose (Sigma-Aldrich) for 1.5 h at 4°C. The resin was washed four times with 1 mL of phosphate-buffered saline, and 40 μL of 2× SDS sample buffer was added, followed by incubation at 95°C for 5 min. The supernatant was then loaded onto a 10% SDS-PAGE gel. For immunoblot analysis, monoclonal anti-HA or anti-c-Myc (Sigma-Aldrich) antibody produced in mice was used as a primary antibody at a 1:2,000 dilution, and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) was employed as a secondary antibody at a dilution of 1:5,000.

Transient Expression Assays with Arabidopsis Protoplasts

The effector plasmids Pro35S:GD:LBD18 and Pro35S:Ω:LBD18 and the reporter plasmids ProGal4(3X):LUC, ProEXP14:LUC, and Pro35S:GUS were described previously (Lee et al., 2013a). LBD18L120P,L127P,V131P,L138P full-length DNA was amplified by PCR from the YFPN:LBD18L120P,L127P,V131P,L138P plasmid using the Pfu DNA polymerase (Solgent) and inserted into the Pro35S:GD:LBD18 vector (Lee et al., 2013a) at the SalI (N terminus) and NotI (C terminus) sites or into the Pro35S:Ω:LBD18 vector (Lee et al., 2013a) at the XbaI (N terminus) and SacI (C terminus) sites as translational fusion after removal of the LBD18 DNA, yielding the Pro35S:GD:LBD18L120P,L127P,V131P,L138P and Pro35S:Ω:LBD18L120P,L127P,V131P,L138P plasmids, respectively. All PCR-amplified DNA sequences were verified by DNA sequencing. The PCR conditions and primer sequences are shown in Supplemental Table S1. These plasmids were purified using the Qiagen Plasmid Midi Kit prior to protoplast transfection. Protoplasts from Arabidopsis plants were prepared and transient gene expression assays were conducted as described previously (Lee et al., 2008). Protoplasts were isolated from the rosette leaves of 2- or 3-week-old Arabidopsis plants under a 16-h photoperiod. The mesophyll protoplasts were transfected with plasmid DNA by polyethylene glycol-mediated protoplast transfection and incubated for 18 h in the dark at 23°C. For transient expression assays, samples were collected in liquid nitrogen, and the total proteins were extracted using 1× passive lysis buffer (Promega) according to the manufacturer’s protocol. LUC activity was then determined using the Dual-Luciferase Reporter Assay System (Promega) with a GloMax Luminometer (Promega). GUS activity was assayed with 1 mm 4-methylumbelliferyl-β-d-glucuronide in GUS extraction buffer. After terminating the reaction with 0.2 m Na2CO3, the appearance of the GUS methylumbelliferyl reaction product was measured with an FLx800 microplate fluorescence reader (Bio-Tek Instruments). LUC activity was normalized to the GUS activity. Transfection was conducted in triplicate. LUC and GUS assays were performed in duplicate for each transfection.

Statistical Analysis

Quantitative data were subjected to statistical analysis for every pairwise comparison using Student’s t test (Predictive Analytics Software for Windows version 20.0).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: LBD16 (At2g42430), LBD18 (At2g45420), AUX1 (At2g38120), LAX3 (At1g77690), PG (At5g14650), CDKA1;1 (At3g48750), CDKB1;1 (At3g54180), CYCB1;1 (At4g37490), EXP14, (At5g56320), and EXP17 (At4g01630).

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Single and double mutational analyses of the coiled-coil motif of LBD16 to examine protein-protein interactions in Arabidopsis mesophyll protoplasts.

  • Supplemental Figure S2. Triple mutational analyses of the coiled-coil motif of LBD16 to examine protein-protein interactions in Arabidopsis mesophyll protoplasts.

  • Supplemental Figure S3. Single and double mutational analyses of the conserved coiled-coil motif of LBD18 to examine protein-protein interactions in Arabidopsis mesophyll protoplasts.

  • Supplemental Figure S4. Triple mutational analyses of the conserved coiled-coil motif of LBD18 to examine protein-protein interactions in Arabidopsis mesophyll protoplasts.

  • Supplemental Figure S5. Western-blot analysis of proteins isolated from Pro35S:HA:LBD16/lbd16, Pro35S:HA:LBD16Q/lbd16, Pro35S:HA:LBD18/lbd18, and Pro35S:HA:LBD18Q/lbd18 Arabidopsis transgenic lines.

  • Supplemental Figure S6. Sequence alignment of the coiled-coil motifs of LBD16 and LBD18 according to the letter designation of the Leu zipper of bZIP.

  • Supplemental Table S1. Oligonucleotides and PCR conditions.

Acknowledgments

We thank Dr. Jörg Kudla for the pUC-SPYNE and pUC-SPYCE vectors, Dr. Soo Young Kim for the pCITE-4a vector, and Dr. Eunae Kim for critical comments on the validation of the structure modeling.

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.17.00013

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jungmook Kim (jungmkim{at}jnu.ac.kr).

  • H.W.L., N.Y.K., S.K.P., and C.C. designed and conducted the experiments and analyzed the data; S.H.L. built and wrote the molecular models of the coiled coils of LBD16 and LBD18; J.K. conceived the research, designed the experiments, analyzed the data, and wrote the article.

  • ↵1 This work was supported by the Next-Generation BioGreen 21 Program (grant no. PJ01104701), Rural Development Administration, Republic of Korea, and the Mid-Career Researcher Program (grant no. 2016R1A2B4015201) through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology of Korea (to J.K.).

Glossary

LOB
lateral organ boundaries
BiFC
bimolecular fluorescence complementation
LCI
firefly luciferase complementation imaging
CaMV
cauliflower mosaic virus
DEX
dexamethasone
bZIP
basic-region Leu zipper
qRT
quantitative reverse transcription
RT
reverse transcription
  • Received January 6, 2017.
  • Accepted March 22, 2017.
  • Published March 23, 2017.

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Dimerization in LBD16 and LBD18 Transcription Factors Is Critical for Lateral Root Formation
Han Woo Lee, Na Young Kang, Shashank K. Pandey, Chuloh Cho, Sung Haeng Lee, Jungmook Kim
Plant Physiology May 2017, 174 (1) 301-311; DOI: 10.1104/pp.17.00013
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