Arabidopsis carboxyl-terminal domain phosphatase-like isoforms share common catalytic and interaction domains but have distinct in planta functions

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.

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.

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.

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.

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.

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 C24_RD29a-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).

Figure 6.

Interaction of BRCT domains from CPL3 and CPL4 with RAP74 C-terminal region. The recombinant GST-RAP74_466-543 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.