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GIP1 may act as a coactivator that enhances transcriptional activity of LBD18 in Arabidopsis
Journal of Plant Physiology 171 (2014) 14–18
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short Communication
GIP1 may act as a coactivator that enhances transcriptional activity of LBD18 in Arabidopsis Han Woo Lee a , Jong Hwa Park b,1 , Moung Yeon Park b , Jungmook Kim a,∗ a b
Department of Bioenergy Science and Technology and Kumho Life Science Laboratory, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Plant Biotechnology, Chonnam National University, Gwangju 500-757, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 September 2013 Received in revised form 7 November 2013 Accepted 7 November 2013 Available online 11 December 2013 Keywords: Arabidopsis Coactivator G-box binding factor (GBF) GBF interacting protein 1 (GIP1) Lateral organ boundaries domain 18 (LBD18)
a b s t r a c t The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes a class of transcription factors harboring a conserved plant-specific lateral organ boundaries domain and plays a key role in lateral organ development of plants. Recent studies have revealed developmental functions of some LBD genes in Arabidopsis, rice, and maize. We have shown previously that LBD18/ASL20 promotes the emergence of lateral roots in Arabidopsis. LBD18 induces EXPANSIN14 (EXP14) expression by binding to a specific region of the EXP14 promoter. To further understand the molecular mechanism of LBD18 acting as a transcription factor, we isolated a protein interacting with LBD18 by screening an Arabidopsis cDNA library using the yeast two-hybrid system with LBD18 as bait. We found that GBF INTERACTING PROTEIN1 (GIP1) interacts with LBD18 in yeast and Arabidopsis protoplasts. Reverse-transcription-polymerase chain reaction analysis showed overlapping expression of GIP1 and LBD18 in various tissues of Arabidopsis such as roots, aerial parts, and rosette leaves. Transient gene expression assay results with Arabidopsis protoplasts indicated that GIP1 enhances transcriptional activity of LBD18 in the EXP14 promoter fused to the GUS reporter gene. These results show that GIP1 may act as a transcriptional coactivator of LBD18. © 2013 Elsevier GmbH. All rights reserved.
Introduction The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) proteins are a unique class of plantspecific transcription factors that play roles in lateral organ development of plants (Husbands et al., 2007). There are 42 members in Arabidopsis, 35 members in rice, 43 members in maize, and 57 members in poplar (Iwakawa et al., 2002; Shuai et al., 2002; Yang et al., 2006; Zhu et al., 2007). All Arabidopsis LBD/ASL proteins are defined by the characteristic N-terminal lateral organ boundaries (LOB) domain (Shuai et al., 2002; Majer and Hochholdinger, 2011). The LOB domain is approximately 100 amino acids in length and contains a conserved four-Cys motif with CX2 CX6 CX3 C spacing, a Gly-Ala-Ser (GAS) block, and a predicted coiled-coil motif with LX6 LX3 LX6 L spacing reminiscent of a Leu-zipper (Shuai et al., 2002). Forty-two Arabidopsis LBD genes are assigned to two classes of LBD
Abbreviations: ASL, asymmetric leaves2-like; ARR7, Arabidopsis response regulator 7; BiFC, bimolecular fluorescence complementation; DEX, dexamethasone; EMSA, electrophoretic mobility shift assay; EXP, expansin; GBF, G-box binding factor; GIP1, GBF interacting protein 1; LBD, lateral organ boundaries domain. ∗ Corresponding author. Tel.: +82 62 530 5187; fax: +82 62 530 5342. E-mail address: [email protected] (J. Kim). 1 Present address: Department of Biology, Institute of Agricultural Sciences, ETH Zurich, Switzerland. 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.11.003
genes that comprise thirty six class I genes and six class II genes (Iwakawa et al., 2002; Shuai et al., 2002; Majer and Hochholdinger, 2011). The class I proteins harbor LOB domains similar to those observed in the LOB protein, whereas the class II proteins are less similar to the class I proteins but share a conserved amino terminus with limited conservation outside the LOB domain. Selection and binding assays have demonstrated that a truncated LOB protein containing only the LOB domain, AS2, and LBD4 preferentially binds unique DNA sequences in an electrophoretic mobility shift assay (EMSA) (Husbands et al., 2007). The conserved proline residue in the LOB domain of LBD18 is critical for DNAbinding and biological function (Lee et al., 2013a). Several studies have demonstrated that the C-terminal region from LBD16, LBD18, LBD30, and the rice LBD protein, adventitious rootless 1, activates reporter gene expression when fused to the Gal4-DNA binding domain in yeast or Arabidopsis protoplasts (Liu et al., 2005; Borghi et al., 2007; Lee et al., 2013b). These results suggest that some LBD proteins act as DNA-binding transcriptional activators. We recently showed that LBD18 activates EXPANSIN14 (EXP14) expression by binding to a specific region of the EXP14 promoter (Lee et al., 2013b). In the present study, we isolated a putative LBD18 transcription coactivator, G-Box binding factor (GBF) interacting protein 1 (GIP1), using a yeast two-hybrid system. We demonstrated that GIP1 interacts with LBD18 in yeast and Arabidopsis protoplasts. Transient coexpression of LBD18 and GIP1 in Arabidopsis protoplasts
H.W. Lee et al. / Journal of Plant Physiology 171 (2014) 14–18
significantly enhanced expression of the GUS reporter gene fused to the EXP14 promoter. These results indicate that GIP1 may play a role as a LBD18 transcriptional coactivator in the EXP14 promoter. Materials and methods Plant material and growth conditions
15
PCR-amplified DNA sequences were used for subcloning after verification by DNA sequencing. PCR conditions and primer sequences are shown in Table S1. These plasmids were purified using a Qiagen Plasmid Midi kit prior to protoplast transfection (Qiagen, Valencia, CA, USA). Protoplasts were isolated from Arabidopsis plants and transient gene expression assays were conducted as described previously (Lee et al., 2013b).
Arabidopsis thaliana (Col-0) (Lehle Seeds, Round Rock, TX, USA) seedlings were grown under a 16-h photoperiod and treated as described previously (Park et al., 2002). Plasmid construction and Arabidopsis transformation The GIP1 coding region was subcloned into pDONRTM 221 (Invitrogen, Carlsbad, CA, USA) using the Gateway® BP recombination reaction to yield pDONR-GIP1. This construct was subcloned into destination vector pB7WG2,0 (bar) (destination vector, VIB, Gent, Belgium) using the Gateway® LR recombination reaction to yield Pro35S :GIP1. Pro35S :GIP1 transgenic Arabidopsis plants were generated by Agrobacterium tumefaciensmediated transformation. Pro35S :LBD18:GR/lbd16lbd18 (female) (Lee et al., 2009) was crossed with Pro35S :GIP1 (male), yielding Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant Arabidopsis. Homozygous lines were isolated by genotyping for Pro35S :GIP1 and Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant plants. All constructs were confirmed by DNA sequencing prior to plant transformation. The primer sequences used in this study are shown in Table S1. Yeast two-hybrid screening LBD18, GIP1, or GIP1L full-length DNA was amplified by polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) and inserted into the pGBT9.BS and pGAD.GH vectors (Kim et al., 1997) at the EcoRI (N-terminus) and SpeI (C-terminus) sites as a translational fusion with Gal4BD or Gal4AD, yielding the Gal4BD:LBD18, Gal4BD:GIP1, Gal4BD:GIP1L, Gal4AD:LBD18, Gal4AD:GIP1, or Gal4AD:GIP1L DNA constructs. PCR conditions and primer sequences are shown in Table S1. Yeast transformation was performed using the PEG/LiAcetate method as described previously (Gietz et al., 1992). Yeast strain Y190 was first transformed with the pGal4BD-LBD18 plasmid followed by transformation with the A. thaliana cDNA library (Kim et al., 1997). Yeast two-hybrid screening and filter-lift assays were performed as described previously (Kim et al., 1997). Reporter and effector plasmids The 35S:GUS, Pro35S :˝:LBD18, and ProEXP14 :LUC plasmids have been described previously (Lee et al., 2013b). The GIP1 and Arabidopsis response regulator 7 (ARR7) full-length DNAs were amplified by PCR using Pfu DNA polymerase (Stratagene) and inserted into the Pro35S :˝:LBD18 plasmid (Lee et al., 2013b) at the SpeI and SacI sites in place of LBD18, yielding the Pro35S :˝:GIP1 and Pro35S :˝:ARR7 constructs. All constructs were verified by DNA sequencing. PCR conditions and primer sequences are shown in Table S1. Transient expression assays with Arabidopsis protoplasts The GIP1 full-length DNA was amplified by PCR using Pfu DNA polymerase (Stratagene) and inserted into the Pro35S :EGFP:LBD18 vector in place of LBD18 (Lee et al., 2009) at XhoI sites as a translational fusion with EGFP, yielding the Pro35S :EGFP:GIP1 construct.
Fig. 1. Interaction between LBD18 and GIP1 or GIP1L in the yeast two-hybrid system. (A) Protein–protein interaction between GIP1 and LBD18. IAA1 was used as the positive control. Growth of yeast transformants was evaluated on SC (+ His), SC-His (− His) containing 25 mM 3-AT, and the colony color of the transformants was determined by the filter-lift assay (X-Gal). (B) Organ-specific expression of GIP1, GIP1L, and LBD18 in Arabidopsis. Total RNA extracted from various organs of Arabidopsis plants was amplified by RT-PCR analysis with GIP1, GIP1L, and LBD18 primers. ACTIN7 was used as the loading control. YS, 10-day-old seedlings; Rt, root parts of 4-week-old seedlings; Lf; rosette leaves from 4-week-old seedlings; Cl, cauline leaves from 4-week-old seedlings; St, stem of 4-week-old seedlings; Fl, flower of 4-week-old seedlings. (C) Protein–protein interaction between GIP1L and LBD18.
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Subcellular localization and bimolecular fluorescence complementation (BIFC) assays LBD18 and GIP1 DNAs were amplified by PCR using Pfu DNA polymerase and inserted into the N- and C-terminal fragments of yellow fluorescence protein (YFPN and YFPC ) vectors (Walter et al., 2004) at the XbaI and XhoI sites as a translational fusion with YFPN or YFPC , yielding the YFPN -LBD18 and YFPC -GIP1 constructs, respectively. PCR conditions and primer sequences are shown in Table S1. The nuclear localization of the GFP-fusion proteins was monitored by capturing green fluorescence protein (GFP) images, and BIFC was monitored by capturing YFP images, with a TCS SP5 AOBS® spectral confocal and multiphoton microscope system (Leica Microsystems, Wetzlar, Germany). Confocal images of the GFP and YFP fusion proteins were acquired at the Korea Basic Science Institute. Argon (488 nm) and helium/neon lasers (633 nm) were used for GFP and YFP excitation and autofluorescence of chlorophyll excitation, respectively.
RNA isolation and RT-PCR analysis Following treatment, Arabidopsis plants were immediately frozen in liquid nitrogen and stored at −80 ◦ C. Total RNA was isolated from frozen Arabidopsis using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA) and an RNeasy Plant Mini kit (Qiagen), and subjected to RT-PCR analysis with the Access RT-PCR System (Promega, Madison, WI, USA), according to the manufacturer’s instructions. RT-PCR conditions and primer sequences are shown in Table S1.
Statistical analysis Quantitative data were subjected to statistical analysis for every pair-wise comparison using the software for Student’s t-test and for a multiple comparison of means using the Tukey’s honestly significant difference test (Predictive Analytics Software for Windows ver. 20.0; SAS Institute, Cary, NC, USA).
Results and discussion Seventy-two putative positive clones were isolated from yeast two-hybrid screening with LBD18 as bait. We focused on GIP1 (At3g13222) to study protein–protein interactions with LBD18, as GIP1 increases DNA-binding activity of GBFs in vitro (Sehnke et al., 2005). To verify the interaction between LBD18 and GIP1, we isolated the full-length GIP1 cDNA from Arabidopsis seedlings. Full-length coding regions of GIP1 and LBD18 were translationally fused to the Gal4 DNA-binding domain (Gal4BD) or Gal4 transcription activation domain (Gal4AD) (Gal4BD-LBD18, Gal4BD-GIP1, Gal4AD-LBD18, and Gal4AD-GIP1), respectively. These constructs were transformed into the yeast reporter strain Y190 harboring the two reporter genes His3 and LacZ. HIS3 reporter activity was assayed by testing growth of the transformants on SC medium lacking histidine followed by a filter-lift assay to monitor activation of the LacZ reporter gene. The yeast strains that contained the Gal4BD-LBD18, Gal4BD-GIP1, Gal4AD-LBD18, or Gal4AD-GIP1 constructs, respectively, did not activate the reporter gene (Fig. 1A). However, the yeast strain containing the Gal4BD-LBD18 and Gal4AD-GIP1 constructs grew on SC-His with 25 mM 3-AT and turned blue in the filter-lift assay (Fig. 1A). These results show that LBD18 and GIP1
Fig. 2. GIP1 interacts with LBD18 in Arabidopsis protoplasts. (A) Nuclear localization of GIP1-EGFP fusion proteins. The nucleus was identified by 4 ,6-diamidino-2phenylindole (DAPI) staining. Pictures represent epifluorescence (GFP), autofluorescence (chloroplast), and merged images of mesophyll protoplasts transfected with various plasmid DNAs. Bars = 10 m. (B) BiFC assay for GIP1 interaction with LBD18 in Arabidopsis protoplasts. Schematic diagrams of LBD18, LBD18P109L , and GIP1 are shown on the left. IAA1 was used as the positive control. Images represent epifluorescence (YFP), autofluorescence (chloroplast), and merged images of mesophyll protoplasts transfected with various plasmid DNAs. Bars = 10 m.
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interact in yeast and that GIP1 does not possess transcriptionactivating capacity alone. A GIP1 homologue (At1g55820), showing 74% amino acid sequence similarity (including 62% identical residues) to GIP1, was identified in the Arabidopsis genome using InterProScanar in the EMBL-EBI program and is referred to as GIP1LIKE (GIP1L) (Fig. S1). Both GIP1 and GIP1L contained an N-terminal DUF1296 domain, which is a plant-specific motif of unknown function. RT-PCR showed that the LBD18, GIP1, and GIP1L transcripts were detected in all tissues examined (Fig. 1B) and showed overlapping expression patterns. We examined the protein interaction between GIP1L and LBD18 using the yeast two-hybrid system. The yeast strain that contained the Gal4BD-LBD18 and Gal4AD-GIP1L did not grow on SC medium lacking histidine and containing 25 mM 3AT and did not turn blue in the filter-lift assay (Fig. 1C). However, the yeast strain harboring Gal4BD-GIP1L grew on SC-His with 25 mM 3-AT and turned blue in the filter-lift assay (Fig. 1C). These results indicate that the GIP1L protein contains a transcription-activating domain unlike GIP1 but cannot interact with LBD18 in yeast. The GIP1 protein is localized in the nucleus in immunolocalization assays (Sehnke et al., 2005). To further confirm that the GIP1 protein is localized in the nucleus, we determined the subcelluar localization of the GIP1-GFP fusion protein in Arabidopsis mesophyll cell protoplasts. As shown in Fig. 2A, the GIP1-GFP fusion protein was preferentially localized in the nucleus, consistent with the immunolocalization assay. We next used the bimolecular fluorescence complementation (BiFC) assay (Walter et al., 2004) to determine the interaction between LBD18 and GIP1 in plant cells. LBD18 was fused to the N-terminal fragment of YFP (YFPN ) to form YFPN -LBD18. GIP1 was fused to the C-terminal fragment of YFP (YFPC ) to form YFPC -GIP1. Arabidopsis mesophyll cell protoplasts were transfected with YFPN -LBD18 and YFPC -GIP1 constructs, and the subcellular localization of the YFP fusion protein was determined by confocal microscopy. A strong YFP signal was detected in the nucleus when YFPN -LBD18 was coexpressed with YFPC GIP1 (Fig. 2B), demonstrating that LBD18 interacts with GIP1 in plant cells. The conserved proline residue in the LOB domain of LBD18 is critical for DNA-binding and biological function (Lee et al., 2013a) and the LOB domain may have some role in protein–protein interactions (Majer et al., 2012). Arabidopsis protoplasts were transfected with YFPN -LBD18P109L to test the role of the LOB domain of LBD18 in protein–protein interactions with GIP1 (Lee et al., 2013a). YFPC -GIP1 constructs were prepared and a confocal microscopic analysis was conducted. The YFP signal was not detectable when YFPN -LBD18P109L was co-expressed with YFPC -GIP1 in Arabidopsis protoplasts (Fig. 2B), showing that the mutation in the conserved Pro residue of the LOB domain abolished the protein–protein interaction between GIP1 and LBD18. We obtained the same result with the yeast two-hybrid assay (Fig. S2). These results indicate that the LOB domain structure of LBD18 is essential for protein–protein interactions between LBD18 and GIP1. GIP1 strongly interacts with GBFs and enhances DNA-binding affinity of GBFs in vitro (Sehnke et al., 2005). LBD18 acts as a DNAbinding transcription factor in the EXP14 promoter, inducing EXP14 expression (Lee et al., 2013b). We employed protoplast cotransfection assays with the reporter plasmid ProEXP14 :LUC, and the effector Pro35S :˝:LBD18 and Pro35S :˝:GIP1 plasmids to investigate whether GIP1 enhances LBD18 transcriptional activity in the EXP14 promoter (Fig. 3A). Pro35S :˝:ARR7 was used as a negative control in place of Pro35S :˝:LBD18. LBD18 enhanced LUC expression of ProEXP14 :LUC two-fold. GIP1 increased LBD18-mediated transcriptional activity by ∼40% but ARR7 did not (Fig. 3B). We showed previously that LBD18 overexpression rescues lateral root formation in lbd16 lbd18 double mutants (Lee et al., 2009). To test if GIP1 could also regulate the biological function of LBD18 during lateral root formation, we overexpressed GIP1 under the control of the constitutive CaMV 35S promoter in the
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Fig. 3. GIP1 enhances LBD18-induced transcriptional activation in the EXP14 promoter of Arabidopsis protoplasts. (A) Schematic diagrams showing effector plasmids and reporter plasmids. The Pro35S :˝:ARR7 effector plasmid was used as the negative control. (B) Transient gene expression assays with Arabidopsis mesophyll protoplasts. Bars represent standard errors. Data are mean ± standard error of three independent experiments. Asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**) using Student’s t-test. Letters above bars indicate statistically significant differences by the Tukey’s honestly significant difference test (P < 0.07).
wild type (Pro35S :GIP1) and Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant Arabidopsis (Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18) (Lee et al., 2009). However, the emerged lateral root densities of the three different lines of Pro35S :GIP1 transgenic plants were similar to those of the wild type (Fig. S3). We also found that the total number of emerged lateral roots of Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18 transgenic mutants treated with dexamethasone (DEX) was similar to that of Pro35S :LBD18:GR/lbd16lbd18 plants treated with DEX (Fig. S4), showing that lateral root formation in Pro35S :LBD18:GR/lbd16 lbd18 plants was not affected by GIP1 overexpression. Transcriptional coactivators increase gene expression by interacting with transcriptional activators that bind to DNA (Näär et al., 2001; McKenna and O’Malley, 2002). In mammals, many different coactivators and their mechanisms involved in transcriptional regulation have been elucidated. For example, cyclic AMP response element binding protein binding protein (CBP/p300) increases gene expression by relaxing the chromatin structure at the gene promoter through intrinsic histone acetyltransferase activity and by bringing the components of the basal transcriptional mechanism including RNA polymerase II to the promoter (Ogryzko et al., 1996; Goodman and Smolik, 2000). Although limited information is available on plant transcription coactivators, growth-regulating factor-interacting factor 1 has been identified as a transcription
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coactivator that regulates leaf growth and morphology in Arabidopsis (Kim and Kende, 2004). Nonexpressor of pathogenesis-related gene 1 interacts with TGA transcription factors, and this interaction stimulates DNA-binding activity of TGA transcription factors to their cognate cis-element, activating expression of defense response genes (Després et al., 2000, 2003; Fan and Dong, 2002; Rochon et al., 2006). In this study, we identified GIP1 as a putative transcriptional coactivator of LBD18. A previous study using EMSA showed that GIP1 increases the apparent DNA-binding affinity of both GBF3a and GBF3b (Sehnke et al., 2005). We showed that LBD18 synergistically enhanced LUC expression of the ProEXP14 :LUC reporter gene when coexpressed with GIP1 in Arabidopsis protoplasts, compared with the absence of GIP1 expression (Fig. 3). These results indicate that GIP1 may enhance DNA-binding affinity of LBD18 to the EXP14 promoter through a protein–protein interaction, thereby resulting in increased LBD18 transcriptional activity. However, the present genetic analysis result showed no significant difference in lateral root formation between Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant plants and Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant plants after DEX treatment (Fig. S4), indicating that GIP1 may not be limiting during transcriptional activation of the EXP14 target gene by LBD18 for lateral root formation. Additional transcriptional coregulators may be necessary for full activation of EXP14 and other direct target genes of LBD18 to facilitate lateral root emergence during lateral root formation in Arabidopsis. Acknowledgements This study was supported by grants from the World Class University project of the Ministry of Science, Education, and Technology of Korea (R31-2009-000-20025-0) and from the Next-Generation BioGreen 21 Program (PJ00949103), Rural Development Administration, Republic of Korea to J. Kim.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.11.003. References Borghi L, Bureau M, Simon R. Plant Cell 2007;19:1795–808. Després C, Chubak C, Rochon A, Clark R, Bethune T, Desveaux D, et al. Plant Cell 2003;15:2181–91. Després C, DeLong C, Glaze S, Liu E, Fobert PR. Plant Cell 2000;12:279–90. Fan W, Dong X. Plant Cell 2002;14:1377–89. Gietz D, St Jean A, Woods RA, Schiestl RH. Nucleic Acids Res 1992;20:1425. Goodman RH, Smolik S. Genes Dev 2000;14:1553–77. Husbands A, Bell EM, Shuai B, Smith HM, Springer PS. Nucleic Acids Res 2007;35:6663–71. Iwakawa H, Ueno Y, Semiarti E, Onouchi H, Kojima S, Tsukaya H, et al. Plant Cell Physiol 2002;43:467–78. Kim JH, Kende H. Proc Natl Acad Sci U S A 2004;101:13374–9. Kim J, Harter K, Theologis A. Proc Natl Acad Sci U S A 1997;94:11786–91. Lee HW, Kim NY, Lee DJ, Kim J. Plant Physiol 2009;151:1377–89. Lee HW, Kim MJ, Park MY, Han KH, Kim J. Mol Plant 2013a;6:1722–5. Lee HW, Kim MJ, Kim NY, Lee SH, Kim J. Plant J 2013b;73:212–24. Liu H, Wang S, Yu X, Yu J, He X, Zhang S, et al. Plant J 2005;43:47–56. Majer C, Hochholdinger F. Trends Plant Sci 2011;16:47–52. Majer C, Xu X, Berendzen KW, Hochholdinger F. Philos Trans R Soc B 2012;367:1542–51. McKenna NJ, O’Malley BW. Endocrinology 2002;143:2461–5. Näär AM, Lemon BD, Tjian R. Annu Rev Biochem 2001;70:475–501. Ogryzko VV, Schiltz L, Russanova V, Howard BH, Nakatani Y. Cell 1996;87: 953–9. Park JY, Kim HJ, Kim J. Plant J 2002;32:669–83. Rochon A, Boyle P, Wignes T, Fobert PR, Després C. Plant Cell 2006;18:3670–85. Sehnke PC, Laughner BJ, Lyerlylinebarger CR, Gurley WB, Ferl RJ. Cell Res 2005;8:567–75. ˜ CG, Springer PS. Plant Physiol 2002;129:747–61. Shuai B, Reynaga-Pena Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, et al. Plant J 2004;40:428–38. Yang Y, Yu X, Wu P. Mol Phylogenet Evol 2006;39:248–62. Zhu QH, Guo AY, Gao G, Zhong YF, Xu M, Huang M, et al. Bioinformatics 2007;23: 1307–8.
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short Communication
GIP1 may act as a coactivator that enhances transcriptional activity of LBD18 in Arabidopsis Han Woo Lee a , Jong Hwa Park b,1 , Moung Yeon Park b , Jungmook Kim a,∗ a b
Department of Bioenergy Science and Technology and Kumho Life Science Laboratory, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Plant Biotechnology, Chonnam National University, Gwangju 500-757, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 September 2013 Received in revised form 7 November 2013 Accepted 7 November 2013 Available online 11 December 2013 Keywords: Arabidopsis Coactivator G-box binding factor (GBF) GBF interacting protein 1 (GIP1) Lateral organ boundaries domain 18 (LBD18)
a b s t r a c t The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes a class of transcription factors harboring a conserved plant-specific lateral organ boundaries domain and plays a key role in lateral organ development of plants. Recent studies have revealed developmental functions of some LBD genes in Arabidopsis, rice, and maize. We have shown previously that LBD18/ASL20 promotes the emergence of lateral roots in Arabidopsis. LBD18 induces EXPANSIN14 (EXP14) expression by binding to a specific region of the EXP14 promoter. To further understand the molecular mechanism of LBD18 acting as a transcription factor, we isolated a protein interacting with LBD18 by screening an Arabidopsis cDNA library using the yeast two-hybrid system with LBD18 as bait. We found that GBF INTERACTING PROTEIN1 (GIP1) interacts with LBD18 in yeast and Arabidopsis protoplasts. Reverse-transcription-polymerase chain reaction analysis showed overlapping expression of GIP1 and LBD18 in various tissues of Arabidopsis such as roots, aerial parts, and rosette leaves. Transient gene expression assay results with Arabidopsis protoplasts indicated that GIP1 enhances transcriptional activity of LBD18 in the EXP14 promoter fused to the GUS reporter gene. These results show that GIP1 may act as a transcriptional coactivator of LBD18. © 2013 Elsevier GmbH. All rights reserved.
Introduction The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) proteins are a unique class of plantspecific transcription factors that play roles in lateral organ development of plants (Husbands et al., 2007). There are 42 members in Arabidopsis, 35 members in rice, 43 members in maize, and 57 members in poplar (Iwakawa et al., 2002; Shuai et al., 2002; Yang et al., 2006; Zhu et al., 2007). All Arabidopsis LBD/ASL proteins are defined by the characteristic N-terminal lateral organ boundaries (LOB) domain (Shuai et al., 2002; Majer and Hochholdinger, 2011). The LOB domain is approximately 100 amino acids in length and contains a conserved four-Cys motif with CX2 CX6 CX3 C spacing, a Gly-Ala-Ser (GAS) block, and a predicted coiled-coil motif with LX6 LX3 LX6 L spacing reminiscent of a Leu-zipper (Shuai et al., 2002). Forty-two Arabidopsis LBD genes are assigned to two classes of LBD
Abbreviations: ASL, asymmetric leaves2-like; ARR7, Arabidopsis response regulator 7; BiFC, bimolecular fluorescence complementation; DEX, dexamethasone; EMSA, electrophoretic mobility shift assay; EXP, expansin; GBF, G-box binding factor; GIP1, GBF interacting protein 1; LBD, lateral organ boundaries domain. ∗ Corresponding author. Tel.: +82 62 530 5187; fax: +82 62 530 5342. E-mail address: [email protected] (J. Kim). 1 Present address: Department of Biology, Institute of Agricultural Sciences, ETH Zurich, Switzerland. 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.11.003
genes that comprise thirty six class I genes and six class II genes (Iwakawa et al., 2002; Shuai et al., 2002; Majer and Hochholdinger, 2011). The class I proteins harbor LOB domains similar to those observed in the LOB protein, whereas the class II proteins are less similar to the class I proteins but share a conserved amino terminus with limited conservation outside the LOB domain. Selection and binding assays have demonstrated that a truncated LOB protein containing only the LOB domain, AS2, and LBD4 preferentially binds unique DNA sequences in an electrophoretic mobility shift assay (EMSA) (Husbands et al., 2007). The conserved proline residue in the LOB domain of LBD18 is critical for DNAbinding and biological function (Lee et al., 2013a). Several studies have demonstrated that the C-terminal region from LBD16, LBD18, LBD30, and the rice LBD protein, adventitious rootless 1, activates reporter gene expression when fused to the Gal4-DNA binding domain in yeast or Arabidopsis protoplasts (Liu et al., 2005; Borghi et al., 2007; Lee et al., 2013b). These results suggest that some LBD proteins act as DNA-binding transcriptional activators. We recently showed that LBD18 activates EXPANSIN14 (EXP14) expression by binding to a specific region of the EXP14 promoter (Lee et al., 2013b). In the present study, we isolated a putative LBD18 transcription coactivator, G-Box binding factor (GBF) interacting protein 1 (GIP1), using a yeast two-hybrid system. We demonstrated that GIP1 interacts with LBD18 in yeast and Arabidopsis protoplasts. Transient coexpression of LBD18 and GIP1 in Arabidopsis protoplasts
H.W. Lee et al. / Journal of Plant Physiology 171 (2014) 14–18
significantly enhanced expression of the GUS reporter gene fused to the EXP14 promoter. These results indicate that GIP1 may play a role as a LBD18 transcriptional coactivator in the EXP14 promoter. Materials and methods Plant material and growth conditions
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PCR-amplified DNA sequences were used for subcloning after verification by DNA sequencing. PCR conditions and primer sequences are shown in Table S1. These plasmids were purified using a Qiagen Plasmid Midi kit prior to protoplast transfection (Qiagen, Valencia, CA, USA). Protoplasts were isolated from Arabidopsis plants and transient gene expression assays were conducted as described previously (Lee et al., 2013b).
Arabidopsis thaliana (Col-0) (Lehle Seeds, Round Rock, TX, USA) seedlings were grown under a 16-h photoperiod and treated as described previously (Park et al., 2002). Plasmid construction and Arabidopsis transformation The GIP1 coding region was subcloned into pDONRTM 221 (Invitrogen, Carlsbad, CA, USA) using the Gateway® BP recombination reaction to yield pDONR-GIP1. This construct was subcloned into destination vector pB7WG2,0 (bar) (destination vector, VIB, Gent, Belgium) using the Gateway® LR recombination reaction to yield Pro35S :GIP1. Pro35S :GIP1 transgenic Arabidopsis plants were generated by Agrobacterium tumefaciensmediated transformation. Pro35S :LBD18:GR/lbd16lbd18 (female) (Lee et al., 2009) was crossed with Pro35S :GIP1 (male), yielding Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant Arabidopsis. Homozygous lines were isolated by genotyping for Pro35S :GIP1 and Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant plants. All constructs were confirmed by DNA sequencing prior to plant transformation. The primer sequences used in this study are shown in Table S1. Yeast two-hybrid screening LBD18, GIP1, or GIP1L full-length DNA was amplified by polymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) and inserted into the pGBT9.BS and pGAD.GH vectors (Kim et al., 1997) at the EcoRI (N-terminus) and SpeI (C-terminus) sites as a translational fusion with Gal4BD or Gal4AD, yielding the Gal4BD:LBD18, Gal4BD:GIP1, Gal4BD:GIP1L, Gal4AD:LBD18, Gal4AD:GIP1, or Gal4AD:GIP1L DNA constructs. PCR conditions and primer sequences are shown in Table S1. Yeast transformation was performed using the PEG/LiAcetate method as described previously (Gietz et al., 1992). Yeast strain Y190 was first transformed with the pGal4BD-LBD18 plasmid followed by transformation with the A. thaliana cDNA library (Kim et al., 1997). Yeast two-hybrid screening and filter-lift assays were performed as described previously (Kim et al., 1997). Reporter and effector plasmids The 35S:GUS, Pro35S :˝:LBD18, and ProEXP14 :LUC plasmids have been described previously (Lee et al., 2013b). The GIP1 and Arabidopsis response regulator 7 (ARR7) full-length DNAs were amplified by PCR using Pfu DNA polymerase (Stratagene) and inserted into the Pro35S :˝:LBD18 plasmid (Lee et al., 2013b) at the SpeI and SacI sites in place of LBD18, yielding the Pro35S :˝:GIP1 and Pro35S :˝:ARR7 constructs. All constructs were verified by DNA sequencing. PCR conditions and primer sequences are shown in Table S1. Transient expression assays with Arabidopsis protoplasts The GIP1 full-length DNA was amplified by PCR using Pfu DNA polymerase (Stratagene) and inserted into the Pro35S :EGFP:LBD18 vector in place of LBD18 (Lee et al., 2009) at XhoI sites as a translational fusion with EGFP, yielding the Pro35S :EGFP:GIP1 construct.
Fig. 1. Interaction between LBD18 and GIP1 or GIP1L in the yeast two-hybrid system. (A) Protein–protein interaction between GIP1 and LBD18. IAA1 was used as the positive control. Growth of yeast transformants was evaluated on SC (+ His), SC-His (− His) containing 25 mM 3-AT, and the colony color of the transformants was determined by the filter-lift assay (X-Gal). (B) Organ-specific expression of GIP1, GIP1L, and LBD18 in Arabidopsis. Total RNA extracted from various organs of Arabidopsis plants was amplified by RT-PCR analysis with GIP1, GIP1L, and LBD18 primers. ACTIN7 was used as the loading control. YS, 10-day-old seedlings; Rt, root parts of 4-week-old seedlings; Lf; rosette leaves from 4-week-old seedlings; Cl, cauline leaves from 4-week-old seedlings; St, stem of 4-week-old seedlings; Fl, flower of 4-week-old seedlings. (C) Protein–protein interaction between GIP1L and LBD18.
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Subcellular localization and bimolecular fluorescence complementation (BIFC) assays LBD18 and GIP1 DNAs were amplified by PCR using Pfu DNA polymerase and inserted into the N- and C-terminal fragments of yellow fluorescence protein (YFPN and YFPC ) vectors (Walter et al., 2004) at the XbaI and XhoI sites as a translational fusion with YFPN or YFPC , yielding the YFPN -LBD18 and YFPC -GIP1 constructs, respectively. PCR conditions and primer sequences are shown in Table S1. The nuclear localization of the GFP-fusion proteins was monitored by capturing green fluorescence protein (GFP) images, and BIFC was monitored by capturing YFP images, with a TCS SP5 AOBS® spectral confocal and multiphoton microscope system (Leica Microsystems, Wetzlar, Germany). Confocal images of the GFP and YFP fusion proteins were acquired at the Korea Basic Science Institute. Argon (488 nm) and helium/neon lasers (633 nm) were used for GFP and YFP excitation and autofluorescence of chlorophyll excitation, respectively.
RNA isolation and RT-PCR analysis Following treatment, Arabidopsis plants were immediately frozen in liquid nitrogen and stored at −80 ◦ C. Total RNA was isolated from frozen Arabidopsis using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA) and an RNeasy Plant Mini kit (Qiagen), and subjected to RT-PCR analysis with the Access RT-PCR System (Promega, Madison, WI, USA), according to the manufacturer’s instructions. RT-PCR conditions and primer sequences are shown in Table S1.
Statistical analysis Quantitative data were subjected to statistical analysis for every pair-wise comparison using the software for Student’s t-test and for a multiple comparison of means using the Tukey’s honestly significant difference test (Predictive Analytics Software for Windows ver. 20.0; SAS Institute, Cary, NC, USA).
Results and discussion Seventy-two putative positive clones were isolated from yeast two-hybrid screening with LBD18 as bait. We focused on GIP1 (At3g13222) to study protein–protein interactions with LBD18, as GIP1 increases DNA-binding activity of GBFs in vitro (Sehnke et al., 2005). To verify the interaction between LBD18 and GIP1, we isolated the full-length GIP1 cDNA from Arabidopsis seedlings. Full-length coding regions of GIP1 and LBD18 were translationally fused to the Gal4 DNA-binding domain (Gal4BD) or Gal4 transcription activation domain (Gal4AD) (Gal4BD-LBD18, Gal4BD-GIP1, Gal4AD-LBD18, and Gal4AD-GIP1), respectively. These constructs were transformed into the yeast reporter strain Y190 harboring the two reporter genes His3 and LacZ. HIS3 reporter activity was assayed by testing growth of the transformants on SC medium lacking histidine followed by a filter-lift assay to monitor activation of the LacZ reporter gene. The yeast strains that contained the Gal4BD-LBD18, Gal4BD-GIP1, Gal4AD-LBD18, or Gal4AD-GIP1 constructs, respectively, did not activate the reporter gene (Fig. 1A). However, the yeast strain containing the Gal4BD-LBD18 and Gal4AD-GIP1 constructs grew on SC-His with 25 mM 3-AT and turned blue in the filter-lift assay (Fig. 1A). These results show that LBD18 and GIP1
Fig. 2. GIP1 interacts with LBD18 in Arabidopsis protoplasts. (A) Nuclear localization of GIP1-EGFP fusion proteins. The nucleus was identified by 4 ,6-diamidino-2phenylindole (DAPI) staining. Pictures represent epifluorescence (GFP), autofluorescence (chloroplast), and merged images of mesophyll protoplasts transfected with various plasmid DNAs. Bars = 10 m. (B) BiFC assay for GIP1 interaction with LBD18 in Arabidopsis protoplasts. Schematic diagrams of LBD18, LBD18P109L , and GIP1 are shown on the left. IAA1 was used as the positive control. Images represent epifluorescence (YFP), autofluorescence (chloroplast), and merged images of mesophyll protoplasts transfected with various plasmid DNAs. Bars = 10 m.
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interact in yeast and that GIP1 does not possess transcriptionactivating capacity alone. A GIP1 homologue (At1g55820), showing 74% amino acid sequence similarity (including 62% identical residues) to GIP1, was identified in the Arabidopsis genome using InterProScanar in the EMBL-EBI program and is referred to as GIP1LIKE (GIP1L) (Fig. S1). Both GIP1 and GIP1L contained an N-terminal DUF1296 domain, which is a plant-specific motif of unknown function. RT-PCR showed that the LBD18, GIP1, and GIP1L transcripts were detected in all tissues examined (Fig. 1B) and showed overlapping expression patterns. We examined the protein interaction between GIP1L and LBD18 using the yeast two-hybrid system. The yeast strain that contained the Gal4BD-LBD18 and Gal4AD-GIP1L did not grow on SC medium lacking histidine and containing 25 mM 3AT and did not turn blue in the filter-lift assay (Fig. 1C). However, the yeast strain harboring Gal4BD-GIP1L grew on SC-His with 25 mM 3-AT and turned blue in the filter-lift assay (Fig. 1C). These results indicate that the GIP1L protein contains a transcription-activating domain unlike GIP1 but cannot interact with LBD18 in yeast. The GIP1 protein is localized in the nucleus in immunolocalization assays (Sehnke et al., 2005). To further confirm that the GIP1 protein is localized in the nucleus, we determined the subcelluar localization of the GIP1-GFP fusion protein in Arabidopsis mesophyll cell protoplasts. As shown in Fig. 2A, the GIP1-GFP fusion protein was preferentially localized in the nucleus, consistent with the immunolocalization assay. We next used the bimolecular fluorescence complementation (BiFC) assay (Walter et al., 2004) to determine the interaction between LBD18 and GIP1 in plant cells. LBD18 was fused to the N-terminal fragment of YFP (YFPN ) to form YFPN -LBD18. GIP1 was fused to the C-terminal fragment of YFP (YFPC ) to form YFPC -GIP1. Arabidopsis mesophyll cell protoplasts were transfected with YFPN -LBD18 and YFPC -GIP1 constructs, and the subcellular localization of the YFP fusion protein was determined by confocal microscopy. A strong YFP signal was detected in the nucleus when YFPN -LBD18 was coexpressed with YFPC GIP1 (Fig. 2B), demonstrating that LBD18 interacts with GIP1 in plant cells. The conserved proline residue in the LOB domain of LBD18 is critical for DNA-binding and biological function (Lee et al., 2013a) and the LOB domain may have some role in protein–protein interactions (Majer et al., 2012). Arabidopsis protoplasts were transfected with YFPN -LBD18P109L to test the role of the LOB domain of LBD18 in protein–protein interactions with GIP1 (Lee et al., 2013a). YFPC -GIP1 constructs were prepared and a confocal microscopic analysis was conducted. The YFP signal was not detectable when YFPN -LBD18P109L was co-expressed with YFPC -GIP1 in Arabidopsis protoplasts (Fig. 2B), showing that the mutation in the conserved Pro residue of the LOB domain abolished the protein–protein interaction between GIP1 and LBD18. We obtained the same result with the yeast two-hybrid assay (Fig. S2). These results indicate that the LOB domain structure of LBD18 is essential for protein–protein interactions between LBD18 and GIP1. GIP1 strongly interacts with GBFs and enhances DNA-binding affinity of GBFs in vitro (Sehnke et al., 2005). LBD18 acts as a DNAbinding transcription factor in the EXP14 promoter, inducing EXP14 expression (Lee et al., 2013b). We employed protoplast cotransfection assays with the reporter plasmid ProEXP14 :LUC, and the effector Pro35S :˝:LBD18 and Pro35S :˝:GIP1 plasmids to investigate whether GIP1 enhances LBD18 transcriptional activity in the EXP14 promoter (Fig. 3A). Pro35S :˝:ARR7 was used as a negative control in place of Pro35S :˝:LBD18. LBD18 enhanced LUC expression of ProEXP14 :LUC two-fold. GIP1 increased LBD18-mediated transcriptional activity by ∼40% but ARR7 did not (Fig. 3B). We showed previously that LBD18 overexpression rescues lateral root formation in lbd16 lbd18 double mutants (Lee et al., 2009). To test if GIP1 could also regulate the biological function of LBD18 during lateral root formation, we overexpressed GIP1 under the control of the constitutive CaMV 35S promoter in the
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Fig. 3. GIP1 enhances LBD18-induced transcriptional activation in the EXP14 promoter of Arabidopsis protoplasts. (A) Schematic diagrams showing effector plasmids and reporter plasmids. The Pro35S :˝:ARR7 effector plasmid was used as the negative control. (B) Transient gene expression assays with Arabidopsis mesophyll protoplasts. Bars represent standard errors. Data are mean ± standard error of three independent experiments. Asterisks denote statistical significance at P < 0.05 (*) and P < 0.01 (**) using Student’s t-test. Letters above bars indicate statistically significant differences by the Tukey’s honestly significant difference test (P < 0.07).
wild type (Pro35S :GIP1) and Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant Arabidopsis (Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18) (Lee et al., 2009). However, the emerged lateral root densities of the three different lines of Pro35S :GIP1 transgenic plants were similar to those of the wild type (Fig. S3). We also found that the total number of emerged lateral roots of Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18 transgenic mutants treated with dexamethasone (DEX) was similar to that of Pro35S :LBD18:GR/lbd16lbd18 plants treated with DEX (Fig. S4), showing that lateral root formation in Pro35S :LBD18:GR/lbd16 lbd18 plants was not affected by GIP1 overexpression. Transcriptional coactivators increase gene expression by interacting with transcriptional activators that bind to DNA (Näär et al., 2001; McKenna and O’Malley, 2002). In mammals, many different coactivators and their mechanisms involved in transcriptional regulation have been elucidated. For example, cyclic AMP response element binding protein binding protein (CBP/p300) increases gene expression by relaxing the chromatin structure at the gene promoter through intrinsic histone acetyltransferase activity and by bringing the components of the basal transcriptional mechanism including RNA polymerase II to the promoter (Ogryzko et al., 1996; Goodman and Smolik, 2000). Although limited information is available on plant transcription coactivators, growth-regulating factor-interacting factor 1 has been identified as a transcription
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coactivator that regulates leaf growth and morphology in Arabidopsis (Kim and Kende, 2004). Nonexpressor of pathogenesis-related gene 1 interacts with TGA transcription factors, and this interaction stimulates DNA-binding activity of TGA transcription factors to their cognate cis-element, activating expression of defense response genes (Després et al., 2000, 2003; Fan and Dong, 2002; Rochon et al., 2006). In this study, we identified GIP1 as a putative transcriptional coactivator of LBD18. A previous study using EMSA showed that GIP1 increases the apparent DNA-binding affinity of both GBF3a and GBF3b (Sehnke et al., 2005). We showed that LBD18 synergistically enhanced LUC expression of the ProEXP14 :LUC reporter gene when coexpressed with GIP1 in Arabidopsis protoplasts, compared with the absence of GIP1 expression (Fig. 3). These results indicate that GIP1 may enhance DNA-binding affinity of LBD18 to the EXP14 promoter through a protein–protein interaction, thereby resulting in increased LBD18 transcriptional activity. However, the present genetic analysis result showed no significant difference in lateral root formation between Pro35S :GIP1/Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant plants and Pro35S :LBD18:GR/lbd16lbd18 transgenic mutant plants after DEX treatment (Fig. S4), indicating that GIP1 may not be limiting during transcriptional activation of the EXP14 target gene by LBD18 for lateral root formation. Additional transcriptional coregulators may be necessary for full activation of EXP14 and other direct target genes of LBD18 to facilitate lateral root emergence during lateral root formation in Arabidopsis. Acknowledgements This study was supported by grants from the World Class University project of the Ministry of Science, Education, and Technology of Korea (R31-2009-000-20025-0) and from the Next-Generation BioGreen 21 Program (PJ00949103), Rural Development Administration, Republic of Korea to J. Kim.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.11.003. References Borghi L, Bureau M, Simon R. Plant Cell 2007;19:1795–808. Després C, Chubak C, Rochon A, Clark R, Bethune T, Desveaux D, et al. Plant Cell 2003;15:2181–91. Després C, DeLong C, Glaze S, Liu E, Fobert PR. Plant Cell 2000;12:279–90. Fan W, Dong X. Plant Cell 2002;14:1377–89. Gietz D, St Jean A, Woods RA, Schiestl RH. Nucleic Acids Res 1992;20:1425. Goodman RH, Smolik S. Genes Dev 2000;14:1553–77. Husbands A, Bell EM, Shuai B, Smith HM, Springer PS. Nucleic Acids Res 2007;35:6663–71. Iwakawa H, Ueno Y, Semiarti E, Onouchi H, Kojima S, Tsukaya H, et al. Plant Cell Physiol 2002;43:467–78. Kim JH, Kende H. Proc Natl Acad Sci U S A 2004;101:13374–9. Kim J, Harter K, Theologis A. Proc Natl Acad Sci U S A 1997;94:11786–91. Lee HW, Kim NY, Lee DJ, Kim J. Plant Physiol 2009;151:1377–89. Lee HW, Kim MJ, Park MY, Han KH, Kim J. Mol Plant 2013a;6:1722–5. Lee HW, Kim MJ, Kim NY, Lee SH, Kim J. Plant J 2013b;73:212–24. Liu H, Wang S, Yu X, Yu J, He X, Zhang S, et al. Plant J 2005;43:47–56. Majer C, Hochholdinger F. Trends Plant Sci 2011;16:47–52. Majer C, Xu X, Berendzen KW, Hochholdinger F. Philos Trans R Soc B 2012;367:1542–51. McKenna NJ, O’Malley BW. Endocrinology 2002;143:2461–5. Näär AM, Lemon BD, Tjian R. Annu Rev Biochem 2001;70:475–501. Ogryzko VV, Schiltz L, Russanova V, Howard BH, Nakatani Y. Cell 1996;87: 953–9. Park JY, Kim HJ, Kim J. Plant J 2002;32:669–83. Rochon A, Boyle P, Wignes T, Fobert PR, Després C. Plant Cell 2006;18:3670–85. Sehnke PC, Laughner BJ, Lyerlylinebarger CR, Gurley WB, Ferl RJ. Cell Res 2005;8:567–75. ˜ CG, Springer PS. Plant Physiol 2002;129:747–61. Shuai B, Reynaga-Pena Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, et al. Plant J 2004;40:428–38. Yang Y, Yu X, Wu P. Mol Phylogenet Evol 2006;39:248–62. Zhu QH, Guo AY, Gao G, Zhong YF, Xu M, Huang M, et al. Bioinformatics 2007;23: 1307–8.