Abstract

We showed previously that the ERF-associated amphiphilic repression (EAR) motif is a plant-specific repression domain that contains the conserved amino acid sequence LXLXL. In this report, we describe the identification of a novel repression domain, L/VR/KLFGVXM/V/L, which is different from known EAR motifs, in B3 DNA-binding domain transcription factors in Arabidopsis. Database analysis revealed that 29 Arabidopsis transcription factors, which included members of the RAV, ARF, Hsf and MYB families, contain the R/KLFGV conserved motif found in the novel repression domain. We demonstrated that factors that contain the R/KLFGV motif, namely, RAV1, RAV2, HsfB1 and HsfB2b, exhibited the repressive activity.

Gene expression in plants is regulated predominantly at the transcriptional level, and numerous transcription factors act as key regulators of various biological processes. Moreover, both activators and repressors of transcriptional also play important roles in the regulation of gene expression. The ERF-associated amphiphilic repression (EAR) motif was identified previously as a repression domain in plants (Ohta et al. 2001). The core sequence of this repression domain consists of only six amino acids, which are sufficient for the repressive activity and which, upon fusion to transcriptional activators, converts them into strong repressors (Hiratsu et al. 2003, Hiratsu et al. 2004). The conserved sequence of the EAR motif in various plant transcription factors is LXLXL (where X can be any amino acid), and it is amphiphilic (Ohta et al. 2001, Hiratsu et al. 2004). The EAR motif has been found in various plant proteins, which include AUX-IAA, BZR1, class II ERF and a number of C2H2 zinc-finger proteins, such as SUPERMAN (Ohta et al. 2001, Hiratsu et al. 2002, Tiwari et al. 2004, He et al. 2005). Thus, the EAR motif is not specific to a small family of transcription factors. In Arabidopsis, approximately 10% of plant transcription factors might be transcriptional repressors and it is likely that the possible extent of the contribution of repressors to the regulation of plant functions might be large (Mitsuda N. and Ohme-Takagi M. in preparation).

We showed recently that AtMYBL2 is an active transcriptional repressor but that its repression domain does not contain the LXLXL motif (Matsui et al. 2008). This observation suggests the presence of unidentified groups of transcriptional repressors with novel repression domains. Identification of these putative repression domains and the isolation of novel families of transcriptional repressors would help to clarify the various mechanisms that are controlled by transcriptional repressors in plants.

It was reported previously that transgenic plants that expressed a chimeric repressor, in which the EAR repression domain (SRDX) was fused to a transcription factor, exhibited phenotypic changes similar to those of plants with the corresponding loss-of-function alleles but unlike those of plants that overexpressed the factor itself (Hiratsu et al. 2003). In contrast, when a transcription factor encodes a repressor, the phenotype of the chimeric repressor plants should be similar to that of plants that overexpress the repressor but different from those with the corresponding loss-of-function alleles, as reported previously for AtMYBL2 (Matsui et al. 2008).

In an attempt to identify novel transcriptional repressors, we compared the phenotypes of chimeric repressor-expressing and transcription factor-overexpressing plants for a variety of transcription factors. We found that plants that ectopically expressed At2g36080 (Pro35S:At2g36080), which encodes a plant-specific B3 DNA-binding domain transcription factor closely related to NGA (Supplementary Fig. S4; Alvarez et al. 2006), had a phenotype similar to that of the corresponding chimeric At2g36080 repressor-expressing lines (Pro35S:At2g36080SRDX; Figs. 1A–C). Both Pro35S:At2g36080SRDX and Pro35S:At2g36080 plants exhibited cotyledon fusion and loss of shoot apical meristem (SAM), and the seedlings had abnormally short roots with long root hairs (Fig. 1B, C). Both of the transgenic lines with a mildly abnormal phenotype had narrow leaves and bushy rosettes (Fig. 1B, C). These observations suggested that the product of At2g36080 might be a repressor. Transient expression assays also showed that the At2G36080 effector fused to the yeast Gal4 DNA-binding domain (GalBD; Pro35S:Gal4DB-At2g36080) reduced the expression of a Pro35S-Gal4:LUC reporter gene (Hiratsu et al. 2002) by 90% in co-bombarded leaves of Arabidopsis (Fig. 1G).

Fig. 1

The isolation of a novel transcriptional repressor and its repression domains. (A) Schematic representation of the At2g36080 constructs and derivative. Ω, translation enhancer sequence derived from tobacco mosaic virus. (B–E) Seedlings and rosettes of Pro35S:2g36080SRDX (B), Pro35S:2g36080 (C), Pro35S:del170-244 (D) and the wild type (E). (F) Schematic representation of the Gal4-LUC reporter gene (Fujimoto et al., 2000) and Gal4DB effectors (Fujimoto et al. 2000). (G) Relative luciferase activities after co-bombardment of Arabidopsis leaves with Gal4DB-fused At2g36080 effectors and the Pro35S:Gal4:LUC reporter gene. The relative activity due to the pUC18 vector was set as 1. Error bars indicate the SD (n =3).

Fig. 1

The isolation of a novel transcriptional repressor and its repression domains. (A) Schematic representation of the At2g36080 constructs and derivative. Ω, translation enhancer sequence derived from tobacco mosaic virus. (B–E) Seedlings and rosettes of Pro35S:2g36080SRDX (B), Pro35S:2g36080 (C), Pro35S:del170-244 (D) and the wild type (E). (F) Schematic representation of the Gal4-LUC reporter gene (Fujimoto et al., 2000) and Gal4DB effectors (Fujimoto et al. 2000). (G) Relative luciferase activities after co-bombardment of Arabidopsis leaves with Gal4DB-fused At2g36080 effectors and the Pro35S:Gal4:LUC reporter gene. The relative activity due to the pUC18 vector was set as 1. Error bars indicate the SD (n =3).

Deletion of the C-terminal 75 amino acids from At2g36080 (1–169: del170-244) abolished the repression activity (Fig. 1G), suggesting that the repressive activity of At2g36080 resides between amino acids 170 and 244. Further transient expression assays revealed that del178-192 no longer had repressive activity when fused to the Gal4DB. Moreover, the short peptide, consisting of amino acid residues 178–192, acted as a strong repression domain, similar to the SRDX repression domain, when fused with Gal4DB in transient expression assays (Fig. 1G). The repressive activity of the region from positions 178 to 192 was also confirmed in a transient expression system with the GAL4GCC:LUC reporter and the 35S:AtERF5 effector (Ohta et al. 2000) (Supplementary Fig. S1). These results indicated that the repressive activity of At2g36080 was due to the peptide of 15 amino acids (GNSKTLRLFGVNMEC), which we designated the B3 repression domain (BRD). This motif is neither categorized as an EAR motif nor has similarity to any other sequences known to correspond to a repression domain.

As reflected in the results of transient expression assays (Fig. 1G), transgenic plants that expressed Pro35S:del170-244 were normal, without the cotyledon fusion and bushy phenotype of Pro35S:At2g36080 plants (Fig. 1C, D). These results indicated that repressive activity is necessary for the induction of an abnormal phenotype by Pro35S:At2G36080 in transgenic plants.

To analyze the repressive activity of BRD (GNSKTLRLFGVNMEC) in plants, we fused the sequence that encodes BRD to the coding region of STM, AG and CUC2 (to generate Pro35S:STMBRD, Pro35S:AGBRD and Pro35S:CUC2BRD, respectively), as well as the corresponding SRDX constructs (Fig. 2A). In the case of the STM gene, all Pro35S:STMBRD T1 seedlings lacked a SAM and resembled Pro35S:STMSRDX and stm mutant seedlings (Fig. 2B, Barton and Poethig 1993). In addition, almost all of the flowers on Pro35S:AGBRD T1 plants resembled those of ag mutants (Fig. 2C), as in the case of Pro35S:AGSRDX (Mitsuda et al. 2006). In the case of CUC2BRD, among 99 T1Pro35S:CUC2BRD seedlings examined, two had a fused cotyledon, 59 seedlings had partially fused cotyledons and the remainder were normal (right panel of Fig. 2B). Similarly, in Pro35S:CUC2SRDX plants, 12 seedlings had fused cotyledons, 51 seedlings had partially fused cotyledons and 49 seedlings had normal cotyledons (Fig. 2D). The frequency of the fused cotyledon phenotype in Pro35S:CUC2BRD was 61.6%, which is almost the same as Pro35S:CUC2SRDX (56.3%). Our results indicated that the BRD of At2g36080 is an active repression domain that can convert a transcriptional activator into a dominant repressor when fused with it and expressed in transgenic plants.

Fig. 2

Repressive activity of the repression domain of At2g36080 (BRD) in transgenic Arabidopsis. (A) Schematic representation of the constructs. Ω indicates the Ω translation enhancer sequence. (B) Seedlings of Pro35S:STM2SRDX (left) and Pro35S:STMBRD (right) plants. (C) Flowers of Pro35S:AGBRD plants. (D) Seedlings of Pro35S:CUC2SRDX (left) and Pro35S:CUC2BRD (right) plants. Bars=0.5mm, except bar in right panel of C=1mm

Fig. 2

Repressive activity of the repression domain of At2g36080 (BRD) in transgenic Arabidopsis. (A) Schematic representation of the constructs. Ω indicates the Ω translation enhancer sequence. (B) Seedlings of Pro35S:STM2SRDX (left) and Pro35S:STMBRD (right) plants. (C) Flowers of Pro35S:AGBRD plants. (D) Seedlings of Pro35S:CUC2SRDX (left) and Pro35S:CUC2BRD (right) plants. Bars=0.5mm, except bar in right panel of C=1mm

Further deletion analysis of the BRD of At2g36080 revealed that the eight amino acids LRLFGVNM acted as a repression domain when fused with the Gal4DB in transient expression assays (Fig. 3A). Replacement of the first leucine residue by alanine (ARLFGVNM) abolished the repressive activity (Fig. 3A). In addition, the methionine residue appeared to be involved in the repressive activity because replacement by alanine (LRLFGVNA) also abolished the activity (Fig. 3A). These results indicated that a short peptide of only eight amino acids was able to act as a repression domain.

Fig. 3

Repressive activity of transcriptional repressors that contain a novel repression domain. (A–E) Relative luciferase activities after co-bombardment of Arabidopsis leaves with the Pro35S:Gal4:LUC reporter gene and Gal4DB-fused effectors for the At2g36080 repression domain (LRLFGVNM: 183/190) or its derivatives (A), At3g11580 or NGA1 (B), RAV1 and RAV2 (C), HsfB1 and HsfB2b (D), and deletion of HsfB1 (E), respectively. The left panel of (E) shows schematic representations of the constructs used for transient expression analysis with effectors for HsfB1 and its deletion Error bars indicate the SD (n =3). The relative activity due to the pUC18 vector was set as 1. Error bars indicate the SD (n =3).

Fig. 3

Repressive activity of transcriptional repressors that contain a novel repression domain. (A–E) Relative luciferase activities after co-bombardment of Arabidopsis leaves with the Pro35S:Gal4:LUC reporter gene and Gal4DB-fused effectors for the At2g36080 repression domain (LRLFGVNM: 183/190) or its derivatives (A), At3g11580 or NGA1 (B), RAV1 and RAV2 (C), HsfB1 and HsfB2b (D), and deletion of HsfB1 (E), respectively. The left panel of (E) shows schematic representations of the constructs used for transient expression analysis with effectors for HsfB1 and its deletion Error bars indicate the SD (n =3). The relative activity due to the pUC18 vector was set as 1. Error bars indicate the SD (n =3).

Analysis of other B3 domain transcription factors similar to At2g36080, namely At3g11580, At5g06250, NGA1, NGA2, NGA3 and NGA4, revealed that these proteins also contain sequences similar to LRLFGVNM (Supplementary Figs. S2, S4A). L/VRLFGVN/DM/L/V is the conserved sequence in these proteins. Transient expression assays revealed that At3g11580 and NGA1 had repressive activity as GAL4DB fusions (Fig. 3B). Moreover, deletion of the C-terminus from At3g11580 (del194-267) abolished the repressive activity (Supplementary Fig. S3), indicating that the repression domain of At3g11580 was in the region that included L/VRLFGVN/DM/L/V. The core sequence of the repression domain that was conserved among other B3 domain members was RLFGV (Table 1 and Supplementary Fig. S2). These results suggest that the members of the B3 family that contain the L/VRLFGVN/DM/L/V motif (Supplementary Fig. S2) are likely to have repressive activity. Although the first leucine and the methionine residue are not perfectly conserved among the possible repression domains found in the B3 transcription factors, the amino acids at these position are always hydrophobic, namely leucine, valine and methionine (Table 1 and Supplementary Fig. S2).

Table 1

Transcription factors with a conserved [RK]LFGV sequence in Arabidopsis

Locus Gene name Family name Sequence Position Rice homolog 
At2g36080  ABI3/VP1 GNSKTLRLFGVNMEC –57 Yes 
At3g11580  ABI3/VP1 GSSRTVRLFGVNLEC –50 Yes 
At5g06250  ABI3/VP1 GSSRTVRLFGVNLEC –49 Yes 
At2g46870 NAG1 ABI3/VP1 TAGKRLRLFGVDMEC –66 Yes 
At1g01030 NAG2 ABI3/VP1 TAGKRLRLFGVNMEC –70 Yes 
At3g61970 NAG3 ABI3/VP1 RGEKRLRLFGVDMEC –80 Yes 
At4g01500 NAG4 ABI3/VP1 STTKKLRLFGVDVEE –68 Yes 
At1g13260 RAV1 AP2/ERF DAGRVLRLFGVNISP –38 Yes 
At1g68840 RAV2, TEM2 AP2/ERF PVQVVVRLFGVDIFN –44 Yes 
At3g25730  AP2/ERF ETGRVMRLFGVDISL –30 Yes 
At1g25560 TEM1 AP2/ERF PVQTVVRLFGVNIFN –44 Yes 
At1g35240  ARF KAVTNFRLFGVSLAI –145  
At1g34310  ARF KTGTNFRLFGVTLDT –132  
At1g34390  ARF KTGTNFRLFGVSLVT –139  
At1g34410  ARF KAGTNFRLFGVTLDT –145  
At1g35520  ARF KAGTNFRLFGVSLAT –132  
At1g35540  ARF NAVASFRLFGVSLAT –145  
At4g36990 AT-HsfB1 Hsf GVGEGLKLFGVWLKG –44 Yes 
At5g62020 AT-HsfB2a Hsf EEEASPRLFGVPIGL –44 Yes 
At4g11660 AT-HsfB2b Hsf GEDLTPRLFGVSIGV –60 Yes 
At2g41690 AT-HsfB3 Hsf EEDEGLKLFGVKLE –4  
At1g46264 AT-HsfB4 Hsf SNMRKTKLFGVSLPS –39 Yes 
At3g16350  MYB GSSSAVKLFGVRLTD –336 Yes 
At5g47390  MYB CPNRGVKLFGVRLTE –338 Yes 
At5g56840  MYB YQTRVVRLFGVHLDT –203 Yes 
At5g61620  MYB VNKASVKLFGVNISS –279 Yes 
At1g30810  JUMONJI ASLTKGKLFGVDLM –4  
At2g34880  JUMONJI QSLSKARLFGVDLN –4  
At2g37650  GRAS RLAAYAKLFGVPFE –205  
Locus Gene name Family name Sequence Position Rice homolog 
At2g36080  ABI3/VP1 GNSKTLRLFGVNMEC –57 Yes 
At3g11580  ABI3/VP1 GSSRTVRLFGVNLEC –50 Yes 
At5g06250  ABI3/VP1 GSSRTVRLFGVNLEC –49 Yes 
At2g46870 NAG1 ABI3/VP1 TAGKRLRLFGVDMEC –66 Yes 
At1g01030 NAG2 ABI3/VP1 TAGKRLRLFGVNMEC –70 Yes 
At3g61970 NAG3 ABI3/VP1 RGEKRLRLFGVDMEC –80 Yes 
At4g01500 NAG4 ABI3/VP1 STTKKLRLFGVDVEE –68 Yes 
At1g13260 RAV1 AP2/ERF DAGRVLRLFGVNISP –38 Yes 
At1g68840 RAV2, TEM2 AP2/ERF PVQVVVRLFGVDIFN –44 Yes 
At3g25730  AP2/ERF ETGRVMRLFGVDISL –30 Yes 
At1g25560 TEM1 AP2/ERF PVQTVVRLFGVNIFN –44 Yes 
At1g35240  ARF KAVTNFRLFGVSLAI –145  
At1g34310  ARF KTGTNFRLFGVTLDT –132  
At1g34390  ARF KTGTNFRLFGVSLVT –139  
At1g34410  ARF KAGTNFRLFGVTLDT –145  
At1g35520  ARF KAGTNFRLFGVSLAT –132  
At1g35540  ARF NAVASFRLFGVSLAT –145  
At4g36990 AT-HsfB1 Hsf GVGEGLKLFGVWLKG –44 Yes 
At5g62020 AT-HsfB2a Hsf EEEASPRLFGVPIGL –44 Yes 
At4g11660 AT-HsfB2b Hsf GEDLTPRLFGVSIGV –60 Yes 
At2g41690 AT-HsfB3 Hsf EEDEGLKLFGVKLE –4  
At1g46264 AT-HsfB4 Hsf SNMRKTKLFGVSLPS –39 Yes 
At3g16350  MYB GSSSAVKLFGVRLTD –336 Yes 
At5g47390  MYB CPNRGVKLFGVRLTE –338 Yes 
At5g56840  MYB YQTRVVRLFGVHLDT –203 Yes 
At5g61620  MYB VNKASVKLFGVNISS –279 Yes 
At1g30810  JUMONJI ASLTKGKLFGVDLM –4  
At2g34880  JUMONJI QSLSKARLFGVDLN –4  
At2g37650  GRAS RLAAYAKLFGVPFE –205  

Position refers to the position of valine in the [RK]LFGV sequence, counted from the C-terminus of each respective protein. ‘Yes’ indicates that the rice homolog of each gene encodes an [RK]LFGV sequence.

A database survey revealed that 42 genes in Arabidopsis include the coding sequence for RLFGV or KLFGV, and 29 of these genes encode transcription factors. The transcription factors containing R/KLFGV are members of the ARF, RAV, Hsf, MYB, JUMONJI and GARP families (Table 1). The rice orthologs, with the exception of those for members of the ARF, JUMONJI and GARP families, also include the R/KLFGV sequence (Table 1), indicating that the domain might be important for the biological function of these transcription factors. In the RAV family, four genes, namely RAV1, RAV2 (TEM2) and the closely related genes TEM1 and At3g25730, include the coding sequence for RLFGV in the C-terminal regions of their products (Supplementary Fig. S4A, Table 1). This sequence is also conserved in RAV1 orthologs from various plants (Supplementary Fig. S4B). Transient expression assays showed that RAV1 and RAV2 had repressive activities as Gal4DB fusions, although the repressive activities of these proteins were not as strong as that of At2g36080 (Fig. 3C). These results suggest that RAV-related genes that encode RLFGV might be genes for repressors. Recently, TEM1 was reported to be a repressor of the FT gene and to bind to its promoter region (Castillejo and Pelaz 2008).

Five genes for Hsfs, namely AT-HsfB1, AT-HsfB2a, AT-HsfB2b, AT-HsfB3 and AT-HsfB4, which encode class B Hsf proteins, include coding regions for R/KLFGV (Supplementary Fig. S5A, Table 1). When fused with the Gal4DB, both HsfB1 and HsfB2b had repressive activity, although the repressive activity of HsfB2b was slightly lower than that of HsfB1 (Fig. 3D). The R/KLFGV sequence is conserved in the orthologs of HsfB1 of various plants (Supplementary Fig. S5B). A short peptide, GEGLKLFGVWL, which includes KLFGV, acted as a repression domain when fused with the Gal4DB in a transient expression assay in Arabidopsis (Fig. 3E).

In this study, we identified a novel repression domain which was different from the known EAR-like motif that we had reported previously (Hiratsu et al. 2004). The R/KLFGV sequence appeared to be conserved in this novel repression domain (Table 1). Mutational analysis indicated that the amino acid immediately before and immediately after RLFGV is important for the repressive activity (Fig. 3A). In each transcription factor in this new group, the amino acid that follows RLFGV is methionine, leucine, valine, phenylalanine or isoleucine, each of which is a hydrophobic amino acid (Table 1). In addition, the amino acid immediately before RLFGV is hydrophobic (Table 1), and hydrophobicity at this position might be necessary for the activity of this repression domain. It seems possible, therefore, that the products of At1g30810, At2g34880 (JUMONJI) and At2g37650 (GRAS) might not be repressors because each has an alanine or glycine residue immediately prior to RLFGV.

Active transcriptional repressors are involved in many aspects of plant growth and development, including phytohormone signaling and organ formation. For example, AUX/IAA, ERF, BZR1 and HAT are regulators of the phytohormone response (Sawa et al. 2002, Tiwari et al. 2004, He et al. 2005); SUP, MYBL2 and NGA1 regulate organ formation and cell identity (Bowman et al. 1992, Alvarez et al. 2006, Matsui et al. 2008); and TEM1 and TEM2 (RAV2) regulate flowering time (Castillejo and Pelaz 2008). It is noteworthy, in this context, that all known transcriptional repressors that have been identified in plants are plant specific and regulate plant-specific phenomena. Identification of novel groups of repressors and analysis of the biological functions of each repressor will help us to clarify details of transcriptional regulation in plants.

Materials and Methods

Arabidopsis thaliana Col-0 plants were grown in soil at 22°C with 16h (long-day condition) of light daily. Transformations were performed with Agrobacterium tumefaciens strain GV3101 by the floral dip method (Clough and Bent 1998).

The protein-coding regions of the genes used in this report were amplified from the A. thaliana cDNA library with appropriate primers (see Supplementary Table S1). Effector plasmids included the Gal4DB-coding region fused to the coding sequence of each gene, in-frame, under control of the cauliflower mosaic virus 35S promoter (−800 to +8; CaMV35S). Synthetic DNA fragments encoding each repression domain (see Supplementary Table 1) were annealed and fused to the Gal4DB-coding region in-frame. The reporter gene 35S-Gal4-TATA-LUC-NOS was described previously (Hiratsu et al. 2002). Pro35S:At2g36080 and Pro35S:At2g36080SRDX were constructed from modified vectors derived from pGreenII0029 (Hellens et al. 2000) and p35SSRDXG (Mitsuda et al. 2006). The DNA fragment encoding BRD (see Supplementary Table 1) was synthesized and introduced into the p35SG vector. The resultant p35SBRD vector was used for construction of Pro35S:CUC2BRD, Pro35S:STMBRD and Pro35S:AGBRD.

Transient expression analysis in Arabidopsis leaves and particle bombardment were performed as described previously (Hiratsu et al. 2002).

Amino acid sequences, collected from the CDS data set (TAIR8_cds_20080412), were aligned, and a phylogenetic tree (Neighbor-Joining tree) was constructed from the resultant alignment by CLUSTAL-W (Chenna et al. 2003).

Supplementary data

Supplementary data are available at PCP online.

Acknowledgments

We thank Yoko Ooi, Atsusi Hirano, Sumiko Takahashi and Manami Watanabe for skilled technical assistance.

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Aux/IAA proteins contain a potent transcriptional repression domain.
Plant Cell
 , 
2004
, vol. 
16
 (pg. 
533
-
543
)

Abbreviations:

    Abbreviations:
  • BRD

    B3 repression domain

  • CaMV

    cauliflower mosaic virus

  • GalDB

    Gal4 DNA-binding domain

  • SAM

    shoot apical meristem