Research ArticleENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Arabidopsis Carboxyl-Terminal Domain Phosphatase-Like Isoforms Share Common Catalytic and Interaction Domains But Have Distinct in Planta Functions

Wooyoung Bang, Sewon Kim, Akihiro Ueda, Meenu Vikram, Daejin Yun, Ray A. Bressan, Paul M. Hasegawa, Jeongdong Bahk, Hisashi Koiwa

Published October 2006. DOI: https://doi.org/10.1104/pp.106.084939

Abstract

An Arabidopsis (Arabidopsis thaliana) multigene family (predicted to be more than 20 members) encodes plant C-terminal domain (CTD) phosphatases that dephosphorylate Ser residues in tandem heptad repeat sequences of the RNA polymerase II C terminus. CTD phosphatase-like (CPL) isoforms 1 and 3 are regulators of osmotic stress and abscisic acid (ABA) signaling. Evidence presented herein indicates that CPL3 and CPL4 are homologs of a prototype CTD phosphatase, FCP1 (TFIIF-interacting CTD-phosphatase). CPL3 and CPL4 contain catalytic FCP1 homology and breast cancer 1 C terminus (BRCT) domains. Recombinant CPL3 and CPL4 interact with AtRAP74, an Arabidopsis ortholog of a FCP1-interacting TFIIF subunit. A CPL3 or CPL4 C-terminal fragment that contains the BRCT domain mediates molecular interaction with AtRAP74. Consistent with their predicted roles in transcriptional regulation, green fluorescent protein fusion proteins of CPL3, CPL4, and RAP74 all localize to the nucleus. cpl3 mutations that eliminate the BRCT or FCP1 homology domain cause ABA hyperactivation of the stress-inducible RD29a promoter, whereas RNAi suppression of CPL4 results in dwarfism and reduced seedling growth. These results indicate CPL3 and CPL4 are a paralogous pair of general transcription regulators with similar biochemical properties, but are required for the distinct developmental and environmental responses. CPL4 is necessary for normal plant growth and thus most orthologous to fungal and metazoan FCP1, whereas CPL3 is an isoform that specifically facilitates ABA signaling.

Transcriptional induction of genes that encode stress tolerance determinants is an integral component of plant adaptation responses to adverse environments (Fowler and Thomashow, 2002; Seki et al., 2002; Maruyama et al., 2004). Responsive to Dehydration (RD) genes are ubiquitous outputs of plant stress signal pathway integration that is activated by low temperature, hyperosmolarity, and the plant hormone abscisic acid (ABA), and are presumed necessary for adaptation (Hasegawa et al., 2000; Viswanathan and Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2005). Promoters of genes comprising the RD transcriptome contain dehydration/cold-responsive and ABA-responsive elements that function with DREB/CBF and AREB/ABF DNA-binding transcription factors, respectively, in signal pathway-mediated activation (Stockinger et al., 1997; Liu et al., 1998; Choi et al., 1999; Uno et al., 2000; Kang et al., 2002). Gain- and loss-of-function studies have established that regulation of the RD transcriptome by these transcription factors is a primary determinant of desiccation and cold tolerance (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Chinnusamy et al., 2003).

Recent studies established that plant stress and ABA signaling are regulated by proteins that facilitate various processes in RNA metabolism, including transcription elongation mediated by RNA polymerase II (Pol II), mRNA maturation and export, chromatin structure modification, and microRNA production (Hugouvieux et al., 2001; Stockinger et al., 2001; Xiong et al., 2001, 2002; Koiwa et al., 2002; Levy et al., 2002; Vlachonasios et al., 2003; Han et al., 2004; Borsani et al., 2005). Analysis of Arabidopsis (Arabidopsis thaliana) mutants that are hyperresponsive to osmotic stresses and ABA has identified a family of CTD phosphatase-like (CPL) genes that negatively regulate stress-responsive gene expression (Koiwa et al., 2002; Xiong et al., 2002). CPL1 and CPL3 were so named because the encoded polypeptides (967 and 1,241 amino acids, respectively) are homologous to the FCP1 family of fungal and metazoan protein Ser phosphatases, which regulate transcription by dephosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of Pol II (Lin et al., 2002b).

The CTD of Arabidopsis Pol II contains 34 tandemly repeated heptads with a consensus sequence of Y1S2P3T4S5P6S7 (Nawrath et al., 1990). Dynamic changes in the CTD phosphoarray are controlled by CTD kinases and phosphatases that have Ser-2 and Ser-5 target specificity (Palancade and Bensaude, 2003). Remodeling of the CTD phosphorylation array accompanies the transition from transcriptional initiation to elongation and controls the recruitment, activity, and egress of the various mRNA-processing complexes (Ho and Shuman, 1999; Komarnitsky et al., 2000; Licatalosi et al., 2002; Ahn et al., 2004). Developmental programs and environmental stresses trigger CTD phosphorylation remodeling (Bellier et al., 1997; Dubois and Bensaude, 1998; Bonnet et al., 1999), which is presumed to be a focal process in the differential transcriptional regulation of determinant gene expression.

The Arabidopsis CTD phosphatase gene family is predicted to be composed of more than 20 members based on domain architecture identified in family member proteins of other eukaryotes (Koiwa et al., 2002). CPL3 and CPL4 belong to the FCP1 (TFIIF-interacting CTD phosphatase 1) family of phosphatases that are established to be transcriptional regulators in fungi and metazoa (Archambault et al., 1997, 1998; Hausmann and Shuman, 2002; Lin et al., 2002b). Genetic evidence indicated that CPL3 is a negative regulator of ABA signaling (Koiwa et al., 2002). CPL3 and CPL4 have a conserved N-terminal FCP1 homology (FCPH) domain that contains the consensus DXDXT metal-dependent phosphatase motif and C-terminal breast cancer-1 carboxyl-terminus (BRCT) domain (Archambault et al., 1997; Hausmann and Shuman, 2002). CPL3 has phosphatase activity, but functionality of the BRCT domain is not established. CPL4 functionality is inferred only from sequence homology to CPL3 and other FCP1 homologs. Here, we report the functional dissection of CPL3 and CPL4 proteins in vitro and in vivo. CPL3 and CPL4 are ubiquitously expressed and targeted to nuclei. Molecular interaction of CPL3 or CPL4 with the RAP74 subunit of TFIIF required the C-terminal region that contains the BRCT domain. Loss-of-function/reduced-function genetic analyses indicated that the BRCT domain is necessary for in vivo function of CPL3 and that CPL4, but not CPL3, is essential for general plant growth. These results establish that these isoforms of a core transcriptional complex have distinct biological functions in plants.

RESULTS

FCP1-Like CTD Phosphatase Complex Components in Arabidopsis

The Arabidopsis genome contains two predicted open reading frames (ORFs), At2g33540 (CPL3) and At5g58000 (CPL4), which encode proteins with FCP1 domains. We determined previously that CPL3 encodes a functional phosphatase that regulates stress-responsive transcription and, to a lesser extent, plant growth and development (Koiwa et al., 2002). Genome annotation (The Arabidopsis Information Resource [TAIR]) indicates that At5g58000 encodes CPL4; however, sequence analysis of expressed sequence tag (EST) clone M74J20STM established that polyadenylation occurs at position 2,647 of At5g58000.1 (Fig. 1A ), upstream of the predicted phosphatase-coding sequence. The presence of several EST sequences corresponding to the phosphatase region (GenBank accession nos. BX831510, BX832589, BX832758, BX830307, and BX831690) indicates that CPL4 is transcribed from the second ORF separated from the upstream ORF encoding M74J20STM.

Figure 1.
Figure 1.

Structure of CPL and RAP74 genes and proteins. A, Schematic drawing of CPL4 (At5g58000) locus. A 2,186-bp 3′ region of annotated At5g58000 encodes CPL4. A separate, upstream ORF is confirmed by sequencing EST clone M74JSTM. White and black boxes indicate exons encoding untranslated regions and protein-coding regions. The position of the transcription start site for CPL4 was determined by 5′ RACE and the initiation codon is indicated by an arrowhead. Predicted CPL4 protein is a 440-amino acid peptide containing FCPH and BRCT domains. B, Structure of new cpl3 alleles identified in the T-DNA insertion population. T-DNA insertion alleles cpl3-3 and cpl3-4 encode proteins truncated at the FCPH and BRCT domains, respectively (top). RT-PCR analysis of CPL3 transcripts (bottom). Transcripts corresponding to base position 614 to 886 of CPL3 ORF were detected in all the genotypes. cpl3-3 and cpl3-4 alleles, but not Columbia-0 (Col-0) wild-type, produce CPL3:T-DNA chimeric transcripts that were detected by CPL3_2603 and LBb1 primers. C, Arabidopsis AtRAP74 is a homolog of the eukaryotic TFIIF large subunit. Schematic drawing of AtRAP74 (top). A striped box and a hatched box indicate the conserved acidic region and C-terminal region, respectively. Alignment of conserved C-terminal regions of RAP74 from Arabidopsis (AAR28013), human (NP_002087), and Drosophila (S30237; bottom). Identical residues are shaded.

Analysis of 5′-RACE products identified the CPL4 transcription start site at base 23,497,005 of chromosome V. The CPL4 ORF encodes a 440-amino acid polypeptide that contains a catalytic FCPH domain and a BRCT domain (Fig. 1A). Sequence identities between CPL3 and CPL4 are 44.2% and 54.3% in the FCPH and BRCT domains, respectively. Rice (Oryza sativa) genes that encode homologs of CPL3 and CPL4 were identified also (Supplemental Fig. S1). OsCPL3 is 664 amino acids in length and lacks the long N terminus of AtCPL3. However, the overlapping sequences are 52.8% identical. Two rice genes encode CPL4. OsCPL4a and OsCPL4b are 536 and 420 amino acid residues, respectively, and have 41.4% and 37.9% amino acid identity to AtCPL4 polypeptide, respectively (Supplemental Fig. S1).

The RAP74 subunit of the general transcription factor TFIIF interacts and activates FCP1 family phosphatases (Archambault et al., 1997, 1998). A single Arabidopsis gene (At4g12610) encodes a human RAP74 homolog based on BLAST analysis. A cDNA clone encoding AtRAP74 was obtained by reverse transcription (RT)-PCR and the nucleotide sequence was identical to the sequence annotation NM_117331 in GenBank. The ORF of AtRAP74 encodes a 543-amino acid peptide (Fig. 1C; Supplemental Fig. S2). Two rice homologs, OsRAP74a and OsRAP74b, were identified (Supplemental Fig. S2). The AtRAP74 N terminus is not highly homologous to the corresponding regions in metazoan and fungal RAP74 orthologs. However, the central region (amino acids 231–370) of AtRAP74 is rich in acidic residues (65%), which is a common feature in other RAP74 proteins (Fig. 1C). The C-terminal 108 amino acids of AtRAP74 (position 466–543) has 22% sequence identity to human and Drosophila RAP74 C-terminal regions that interact with cognate FCP1 homologs (Archambault et al., 1997).

CPL3, CPL4, and AtRAP74 were constitutively expressed in both shoots and roots (Fig. 2 ). CPL3 only was induced moderately by NaCl treatment, which may be linked to its role in stress-responsive signaling. CPL3, CPL4, and AtRAP74 green fluorescent protein (GFP) fusion proteins were localized exclusively to the nucleus (Fig. 3 ). The GFP signal was detected within 16 h and nuclear localization did not change throughout the duration of the experiment (3 d).

Figure 2.
Figure 2.

Constitutive and inducible expression of CPL3, CPL4, and RAP74. One-week-old Arabidopsis plants were transferred onto medium with or without 160 mm NaCl and kept for 4 d prior to total RNA extraction. Transcript level for CPL3, CPL4, RAP74, RD29a, and ACT2 genes in shoots and roots were determined by RT-PCR, using 10 μg of total RNA for the RT reaction.

Figure 3.
Figure 3.

CPL3, CPL4, and RAP74 localize in the nucleus of Arabidopsis. A, Ten micrograms of GFP and red fluorescent protein (RFP)-NLS plasmid were introduced into Arabidopsis protoplasts using the polyethylene glycol method. Three days after transformation, cytoplasmic GFP signal (left) and nuclear RFP signal (right) were observed under a fluorescent microscope. B, Ten micrograms of GFP-CPL3, GFP-CPL4, or GFP-RAP74 plasmid were transformed with RFP-NLS into Arabidopsis protoplasts and their subcellular localization was determined as in A. Bars = 10 μm.

CPL4 Facilitates Arabidopsis Growth and Development

Only a T-DNA insertion in the CPL4 5′-untranslated region (cpl4-1: Salk_132900) could be identified and plants lacked any visible phenotypic abnormalities (data not shown). Therefore, RNAi suppression was used to generate CPL4 reduced expression lines. Fifty-five hygromycin-resistant plants were recovered from two independent transformation experiments and these exhibited a range of phenotypic abnormalities. Germination and seedling establishment comparisons were used to categorize the lines into two classes. Eight independent lines (class I) were normal, whereas the rest (class II) exhibited incomplete to no cotyledon expansion (Fig. 4A, b, c, and i–k ). Class II plants grew very slowly and the initial true leaves remained small, had short petioles, and curled downward after the subsequent growth (Fig. 4A, d–g). A group of class II plants (17 lines) subsequently produced leaves that had longer petioles (Fig. 4A, d and e), and developed similarly to wild type. Twenty lines from class II plants matured without recovery from the growth defects and produced small inflorescences (Fig. 4, A and B, f–h). Ten class II lines produced only three to four true leaves in 30 d and did not reach maturity (Fig. 4A, i–k). Hygromycin-resistant vector control lines (25 lines) did not exhibit any of these anomalies (Fig. 4, A and B, a). Cosegregation of the growth phenotype and hygromycin resistance in viable class II RNAi (CPL4) lines was confirmed at the T2 generation (Table I ). All T2 plants with class II phenotype exhibited hygromycin resistance, whereas all plants with wild-type phenotypes were hygromycin sensitive. These results established that class II growth and developmental phenotypes are linked to the RNAi (CPL4) transgene and make it likely that severe growth defects of some class II plants are due to greater suppression of CPL4 expression in these lines.

Figure 4.
Figure 4.

Silencing of CPL4 by RNAi caused severe growth inhibition of transformants. A, Photograph of 1-week-old (a to c) and 1-month-old (d to k) transgenic plants transformed with a vector (pFGC1008, a) or an RNAi (CPL4) construct (pFGCCPL4, b to k) grown in vitro. RNAi (CPL4) plants (class II) failed to expand cotyledons after germination (b and c). d and e, Class II plants that start to show elongation of leaf petioles. f to h, Class II plants that have small curled leaves with short petioles. i to k, Class II plants that show senesced cotyledons and unexpanded true leaves. B, Photograph of 1-month-old, flowering-stage transgenic plants grown on soil. Bars = 1 mm (A) and 10 mm (B), respectively.

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Table I.

Segregation analysis of RNAi (CPL4) plants in T2 generation

Approximately 100 T2 seeds/line were germinated on 1/4× Murashige and Skoog medium and scored for wild-type/class II phenotype. The seedlings were then transferred to 1/4× Murashige and Skoog medium containing 20 μg/mL hygromycin and scored for the hygromycin-resistant (HygR)/sensitive (HygS) phenotype.

The BRCT Domain Is Essential for CPL3 in Vivo Function

CPL3 and CPL4 are distinct CPL isoforms by the presence of the BRCT domain in the C terminus, which is an essential module of metazoan and fungal FCP1 orthologs and is required for catalytic activity of Schizosaccharomyces pombe Fcp1. This contrasts with the human small CTD phosphatase 1 (SCP1) family and Arabidopsis CPL1 and CPL2 that require only the FCPH domain for CTD phosphatase activity. To assess the in planta function of the BRCT domain in CPL3, the capacity of cpl3-3 or cpl3-4 to suppress the ABA hyperinduction of RD29a-luciferase (LUC) expression caused by cpl3-1 (Koiwa et al., 2002) was assessed. The cpl3-1 T-DNA insertion disrupts the CPL3 catalytic domain. The T-DNA insertion in cpl3-3 is at position 3,808 from the ATG initiation codon of genomic sequence At2g33540 and the insertion in cpl3-4 is at position 4,345. cpl3-3 and cpl3-4 alleles resulted in the production of chimeric transcripts that could produce proteins that are truncated in the FCPH and BRCT domains, respectively (Fig. 1B). ABA-inducible expression of RD29a-LUC in F1 plants was determined by CCD image analysis (Koiwa et al., 2002). As shown in Figure 5 , both alleles failed to complement the ABA hyperresponsiveness caused by cpl3-1 (i.e. all alleles are equally dysfunctional). Apparently, the 33-amino acid distal region of the BRCT domain is essential for full biological functionality of CPL3. The CPL3 BRCT domain does not include an evident nuclear localization sequence (NLS) as in the yeast (Saccharomyces cerevisiae) FCP1 (Kops et al., 2002); consequently, this domain in the plant protein apparently does not function to facilitate nuclear localization.

Figure 5.
Figure 5.

CCD imaging analysis of RD29a-LUC reporter gene expression in various cpl3 mutant alleles. The stigmas from Col-0 cpl3-3 and Col-0 cpl3-4 were pollinated with original C24RD29a-LUC cpl3-1 plants. F1 plants were grown for 10 d on 1× Murashige and Skoog, 3% Suc, and 0.7% agar media. LUC image was taken 3 h after spraying 100 μm ABA (A) and relative luminescence intensity from each genotype was quantified using WinView software (B).

BRCT Domains of CPL3 and CPL4 Physically Interact with the RAP74 C Terminus

Yeast and human FCP1 contain a central acidic-hydrophobic region and the BRCT domain distal to the FCPH domain, and this peptide region overlaps with the RAP74-binding region (Archambault et al., 1998). CPL3 and CPL4 catalytic FCPH domains are located in close proximity to the C terminus and the BRCT domain in each protein can be identified on the distal side (Fig. 1). To determine whether the BRCT-containing C-terminal region of CPL3 or CPL4 mediates interaction between CPL and AtRAP74, an immobilized AtRAP74 C-terminal region (77 amino acids) and radiolabeled CPL3 and CPL4 peptide fragments were used to assess in vitro binding. The C-terminal BRCT domain of either CPL3 (amino acids 1,110–1,241) or CPL4 (amino acids 297–440) specifically interacted with immobilized glutathione S-transferase (GST)-RAP74466-543 (Fig. 6 ). A strong interaction is indicated because a significant portion of the labeled CPL peptides remained associated with RAP74 after elution with 0.2 m NaCl. These results establish that the C-terminal BRCT-containing region of CPL3 and CPL4 can associate with the AtRAP74 C-terminal region, which may mediate interaction between CPL and Pol II.

Figure 6.
Figure 6.

Interaction of BRCT domains from CPL3 and CPL4 with RAP74 C-terminal region. The recombinant GST-RAP74466-543 and GST were immobilized on glutathione sepharose and were incubated with ACB buffer containing in vitro synthesized, radiolabeled BRCT domain peptides of CPL3 or CPL4. The unbound peptides were washed with ACB buffer. Bound peptides were sequentially eluted by 0.2 m NaCl and reduced glutathione. One-hundredth input solution (input) and bound peptides in each elution were resolved by tricine SDS-PAGE, and detected by phosphor imager.

DISCUSSION

The Arabidopsis genome encodes a paralogous pair of FCP1 homologs, CPL3 and CPL4, unlike those of fungi and metazoans that have only one. Genetic and biochemical results presented herein establish and differentiate the functions of CPL3 and CPL4. Both proteins localize to the nucleus and interact with RAP74 through a peptide region that includes the BRCT domain. Because RAP74 is a TFIIF subunit that is necessary for the interaction between Pol II and FCP1 (Archambault et al., 1997, 1998), these results indicate CPL3 and CPL4 have functions that are similar to other FCP1 proteins. However, gene suppression experiments revealed that CPL3 and CPL4 have distinct biological functions. cpl3 alleles that disrupt either a catalytic FCPH domain or a BRCT domain cause ABA and NaCl hyperresponsive gene expression, but have moderate effects on plant growth and development. RNAi suppression of CPL4 expression results in growth defects that range from transient retardation of seedling development to lethality prior to maturation. Presumably, the severity of the growth defect is caused by the level of reduced gene expression. This notion is supported indirectly by the fact that T-DNA insertional gene knockout mutations were not identified in any publicly available population. Therefore, it appears that CPL4 may be essential despite sharing overlapping functions with CPL3. This contrasts with another paralogous pair, CPL1 and CPL2, which shares redundant essential functions. Having paralogous FCP1 homologs like CPL3 and CPL4 appears to be ubiquitous in plants because homologs for both CPL3 and CPL4 are present in the rice genome sequence and in EST sequences from various plants, including cotton (Gossypium hirsutum), tobacco (Nicotiana tabacum), and grape (Vitis shuttleworthii). Of these, CPL4 appears to be orthologous to known FCP1 based on the domain structure and its essential function. CPL3 homologs, on the other hand, may function in transducing specific environmental and developmental signals, such as ABA response. Consistent with this hypothesis, a CPL3 homolog of tomato (Lycopersicon esculentum) is a candidate gene for a jointless2 locus that has very specific developmental function, namely, the formation of an abscission zone in fruits (Yang et al., 2005).

CPL3 is the largest known FCP1-like protein (1,242 amino acids), which is attributable to a long N-terminal region preceding the FCPH domain. Located in the CPL3 N terminus is a short region that exhibits homology to yeast Ces1, a multicopy suppressor of cgl1 mutations of the mRNA-capping enzyme gene (Scwer et al., 1998; Koiwa et al., 2002). CPL4 is only 29 amino acids longer than the smallest FCP1 ortholog, which was identified in the microsporidian parasite Encephalitozoon cuniculi (Hausmann et al., 2004). This structural diversity may be indicative of different kinetic properties or protein-protein interaction specificity. CPL3 and CPL4 belong to the FCP1 family that requires Pol II holoenzyme and RAP74 for its CTD phosphatase activity (Archambault et al., 1997, 1998; Kobor et al., 1999). Because strict substrate specificity exists between FCP1 homologs and Pol II phospho-CTD (Lin et al., 2002a; Yeo et al., 2003), further biochemical characterization of CPL3 and CPL4 may require the use of a holocomplex involving at least intact Pol II and AtRAP74.

How do CPL3 and CPL4 (and other CPLs [Yang et al., 2005]) differentially regulate the transcriptome? Recent studies on metazoans indicate that CTD phosphatases interact with specific transcription factors, a process by which the phosphatase is recruited to target gene promoters to regulate expression. For example, Drosophila and human SCPs are recruited specifically by the zinc-finger transcriptional repressor REST/NRSF complex to silence neuronal genes in nonneuronal cells (Yeo et al., 2005). The CPL enzyme, Drosophila eyes absent (Eya), interacts with a homeodomain transcription factor sine oculis and activates expression of genes that are necessary for eye formation. A CTD phosphatase that is recruited to a specific promoter region in this manner may regulate transcription by dephosphorylating Pol II CTD that is phosphorylated by TFIIH (Yeo et al., 2003) or the transcription factor associated with specific target genes (Li et al., 2003; Tootle et al., 2003). By analogy, CPL3 that specifically regulates ABA signaling may be recruited to ABA-responsive promoters by transcription factors and fine-tuned transcription. Potential partners/substrates of CPL3 besides Pol II CTD include zinc-finger transcription factors ABF/AREB that are regulated by phosphorylation/dephosphorylation during ABA-responsive transcription or the homeodomain transcription factor HOS9 and MYB-domain transcription factor HOS10, which were identified in the same mutational screen as cpl3-1 (Zhu et al., 2004, 2005).

Interaction between CPL3/CPL4 and AtRAP74 indicates that both isoforms likely associate with the elongating Pol II complex. Differential regulation of gene expression by CPL isoforms, therefore, may occur also at the level of mRNA elongation and processing. Fine tuning of the CTD phosphoarray in the elongation complex may affect recruitment of RNA-processing factors and production of mature transcripts. Splicing factors such as STABILIZED (Lee et al., 2006) and FCA (Razem et al., 2006) that are involved in osmotic stress or ABA signaling potentially may be some of the recruited regulators. Indeed, FCA interacts with FY, an Arabidopsis homolog of a CTD-binding protein CstF50 (Simpson et al., 2003; Razem et al., 2006). The presence of an ABA-specific CTD phosphatase CPL3 strongly supports the notion that a specific CTD phosphoarray is required to define the signature of the transcriptional output during ABA response.

MATERIALS AND METHODS

Materials

Arabidopsis (Arabidopsis thaliana) plants for protoplast preparation were grown on agar plates containing 1/3× Murashige and Skoog salts and 0.5% Suc. The salt treatment of Arabidopsis seedlings was conducted as described (Koiwa et al., 2003). The pENSOTG GFP fusion vector has been prepared previously (Koiwa et al., 2004). pUCNLSDsRed was provided by Dr. I. Hwang (Jin et al., 2001). Primer sequences used in this research are listed in Table II .

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Table II.

Sequences of primers used in this study

cDNA Cloning and Preparation of GFP Expression Constructs

The transcription start site for CPL4 was determined by 5′-RACE analysis using the 5′-RACE full core set (TaKaRa). Full-length ORF sequences of CPL3, CPL4, and AtRAP74 cDNAs were amplified by PCR (for CPL3) or RT-PCR with primer sets CPL3F and CPL3R, CPL4F and CPL4R, and RAP74F and RAP74R, respectively, then cloned in pET44a between BamHI-NotI sites. For protoplast transformation, the SmaI-NotI fragment of CPL3, CPL4, and RAP74 was excised from pET44a-CPL and pETRAP74 plasmids and inserted in pENSOTG, resulting in pENCPL3, pENCPL4, and pENRAP74.

Expression Analysis by RT-PCR

Total RNA was isolated from Arabidopsis seedlings using the RNeasy plant mini kit (Qiagen). RT-PCR was performed as described previously (Koiwa et al., 2003) using primer sets CPL3RTF and CPL3RTR, CPL4RTF and CPL4RTR, and RAP74CF2 and RAP74CR2. RD29a and ACT2 primers were used as positive controls (Koiwa et al., 2003). For analysis of truncated CPL3 transcripts in cpl3-3 and cpl3-4, RT-PCR was performed using primer pairs CPL3_614 and CPL3_886 for the intact 5′ region and CPL3_2603 and LBb1 for chimeric transcripts.

Protoplast Purification and Transformation

Isolation and transformation of Arabidopsis protoplasts with pENCPL3, pENCPL4, and pENRAP74 plasmids were conducted as described (Koiwa et al., 2004). Transformed protoplasts were incubated at 22°C in the dark. Expression of the fusion constructs was evaluated at 2 and 3 d after transformation as described (Koiwa et al., 2004).

RNAi Analysis

The RNAi construct was prepared by inserting the CPL4 cDNA fragment (position 315–849 bp) into pFGC1008 (http://www.chromdb.org). Resulting pFGCCPL4 was transformed into Agrobacterium tumefaciens GV3101 and was used for flower transformation of Arabidopsis. Transgenic plants were selected on media containing 1/4× Murashige and Skoog salts and 20 μg/mL hygromycin B and 100 μg/mL cefotaxim.

Cosegregation of the transgene and growth phenotype was performed using T2 progeny of viable RNAi (CPL4) lines. Six independent class II lines that contain single-copy T-DNA were used for the analysis. Approximately 100 seeds were grown on media containing 1/4× Murashige and Skoog salts and 0.8% agar for 1 week. The growth phenotype was scored for each plant and then the entire population was transferred to the same media containing 20 μg/mL hygromycin B. Resistance to hygromycin B was scored 5 d after the transfer.

Genetic Analysis

cpl3-3 and cpl3-4 stigmas were pollinated with cpl3-1pollens that contain the RD29a-LUC reporter gene along with a mutated CPL3 allele. ABA-inducible expression of the LUC reporter gene was analyzed in F1 plants as described (Koiwa et al., 2002).

Pull-Down Assay

A cDNA fragment encoding an AtRAP74 C-terminal fragment, RAP74466-543, was amplified by RT-PCR with primers RAP74CF1 and RAP74CR1 and was inserted into the SmaI site of pGEX-2T (GE Healthcare). Expression of GST fusion proteins or control GST protein in Escherichia coli was induced by 0.4 mm isopropylthio-β-galactoside at 37°C for 4 h, and cells were harvested by centrifugation. Cells were resuspended in Tris-buffered saline (10 mm Tris, 0.9% NaCl) and disrupted by sonication. Crude lysate containing approximately 40 μg GST fusion protein was loaded onto a microspin GST column (Amersham) and unbound protein was removed by washing the beads in Tris-buffered saline. The beads were blocked with 3% bovine serum albumin in ACB buffer (10 mm HEPES, pH 7.9, 1 mm EDTA, 1 mm DTT, 10% glycerol) at 4°C for 1 h and washed with ACB wash buffer (ACB buffer containing 0.1 m NaCl; Archambault et al., 1997).

Radiolabeled CPL peptides were prepared using the coupled in vitro transcription/translation system (Promega). cDNA fragments corresponding to the BRCT domain of CPL3 and CPL4 were amplified by two rounds of PCR (first round: M-CPL3, T7-CPL3; M-CPL4, T7-CPL4; second round: M-TAG, T7-TERM). The resulting PCR products contain a T7 promoter sequence and initiation codon followed by an M-tag (MEMMTMSSMM) at the 5′ end, and a T7 terminator sequence at the 3′ end of CPL BRCT domains. The M-tag sequence was used to increase the labeling efficiency by [S35]-Met. PCR products were purified from agarose gel and used as templates for in vitro transcription/translation according to the manufacturer's protocol.

Ten microliters of in vitro translation products were diluted with 40 μL of ACB buffer and added to the glutathione-agarose beads loaded with GST-RAP74 or GST by itself at 4°C for 30 min. After washing with ACB wash buffer (Archambault et al., 1997), bound peptides were eluted by 100 μL 0.2 m NaCl and then 10 mm reduced glutathione, resolved by 12% tricine SDS-PAGE (Schägger and von Jagow, 1987), and were detected by phosphor imager (Molecular Dynamics).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF486633 (AtCPL3), DQ503426 (AtCPL4), ABA93957 (OsCPL3), AAS86390 (OsCPL4a), XP_468260 (OsCPL4b), NM117331 (AtRAP74), AAX95774 (OsRAP74a), AK073337 (OsRAP74b), NP_002087 (human RAP74), and S30237 (Drosophila RAP74).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. Amino acid sequence comparison of Arabidopsis and rice CPL3 and CPL4 homologs.

  • Supplemental Figure S2. Amino acid sequence comparison of Arabidopsis and rice RAP74 homologs.

Acknowledgments

We thank Dr. David Stelly for his assistance in microscopy, Dr. Keyan Zhu-Salzman for her assistance in CCD imaging, and the Arabidopsis Biological Resource Center and the Salk Institute for the SIGnAL mutant collection and for providing T-DNA insertion lines and EST clones.

Footnotes

  • 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: Hisashi Koiwa (koiwa{at}neo.tamu.edu).

  • www.plantphysiol.org/cgi/doi/10.1104/pp.106.084939

  • ↵1 This work was supported by the National Science Foundation (grant nos. DBI–9813360 and MCB0421889); the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service (grant no. 2005–34402–16401 “Designing food for health”); the BK21 Program; and the Environmental Biotechnology National Core Research Center, Gyeongsang National University (grant no. R15–2003–012–01001–0).

  • ↵2 These authors contributed equally to the paper.

  • ↵[W] The online version of this article contains Web-only data.

  • Received June 9, 2006.
  • Accepted August 2, 2006.
  • Published August 11, 2006.

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Arabidopsis Carboxyl-Terminal Domain Phosphatase-Like Isoforms Share Common Catalytic and Interaction Domains But Have Distinct in Planta Functions
Wooyoung Bang, Sewon Kim, Akihiro Ueda, Meenu Vikram, Daejin Yun, Ray A. Bressan, Paul M. Hasegawa, Jeongdong Bahk, Hisashi Koiwa
Plant Physiology Oct 2006, 142 (2) 586-594; DOI: 10.1104/pp.106.084939
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