Plant Mol Biol (2014) 85:443–458 DOI 10.1007/s11103-014-0196-7

Divergence of the expression and subcellular localization of CCR4‑associated factor 1 (CAF1) deadenylase proteins in Oryza sativa Wei‑Lun Chou · Li‑Fen Huang · Jhen‑Cheng Fang · Ching‑Hui Yeh · Chwan‑Yang Hong · Shaw‑Jye Wu · Chung‑An Lu 

Received: 18 November 2013 / Accepted: 25 April 2014 / Published online: 8 May 2014 © Springer Science+Business Media Dordrecht 2014

Abstract  Deadenylation, also called poly(A) tail shortening, is the first, rate-limiting step in the general cytoplasmic mRNA degradation in eukaryotic cells. The CCR4-NOT complex, containing the two key components carbon catabolite repressor 4 (CCR4) and CCR4-associated factor 1 (CAF1), is a major player in deadenylation. CAF1 belongs to the RNase D group in the DEDD superfamily, and is a protein conserved through evolution from yeast to humans and plants. Every higher plant, including Arabidopsis and rice, contains a CAF1 multigene family. In this study, we identified and cloned four OsCAF1 genes (OsCAF1A, OsCAF1B, OsCAF1G, and OsCAF1H) from rice. Four recombinant OsCAF1 proteins, rOsCAF1A, rOsCAF1B, rOsCAF1G, and rOsCAF1H, all exhibited 3′–5′ exonuclease activity in vitro. Point mutations in the catalytic residues of each analyzed recombinant OsCAF1 proteins were shown to disrupt deadenylase activity. OsCAF1A and OsCAF1G mRNA were found to be abundant in the leaves of mature plants. Two types of OsCAF1B mRNA transcript were detected in an inverse expression pattern in various

Electronic supplementary material  The online version of this article (doi:10.1007/s11103-014-0196-7) contains supplementary material, which is available to authorized users. W.-L. Chou · J.-C. Fang · C.-H. Yeh · S.-J. Wu · C.-A. Lu (*)  Department of Life Sciences, National Central University, Jhongli City, Taoyuan County 320, Taiwan, ROC e-mail: [email protected] L.-F. Huang  Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Jhongli City, Taoyuan County 320, Taiwan, ROC C.-Y. Hong  Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan, ROC

tissues. OsCAF1B was transient, induced by drought, cold, abscisic acid, and wounding treatments. OsCAF1H mRNA was not detected either under normal conditions or during most stress treatments, but only accumulated during heat stress. Four OsCAF1-reporter fusion proteins were localized in both the cytoplasm and nucleus. In addition, when green fluorescent protein fused with OsCAF1B, OsCAF1G, and OsCAF1H, respectively, fluorescent spots were observed in the nucleolus. OsCAF1B fluorescent fusion proteins were located in discrete cytoplasmic foci and fibers. We present evidences that OsCAF1B colocalizes with AtXRN4, a processing body marker, and AtKSS12, a microtubules maker, indicating that OsCAF1B is a component of the plant P-body and associate with microtubules. Our findings provide biochemical evidence that OsCAF1 proteins may be involved in the deadenylation in rice. The unique expression patterns of each OsCAF1 were observed in various tissues when undergoing abiotic stress treatments, implying that each CAF1 gene in rice plays a specific role in the development and stress response of a plant. Keywords  CCR4-associated factor 1 (CAF1) · Deadenylase · Stress response · Subcellular localization · Processing body · Oryza sativa

Introduction Higher land plants are sessile and require complex yet coordinated gene activities to withstand various stresses. Gene expression in such plants rapidly changes in response to biotic and abiotic stress, and this occurs not only through transcriptional control by mRNA synthesis (Agarwal et al. 2006; Lata and Prasad 2011; Nakaminami et al. 2012; Nakashima et al. 2009; Puranik et al. 2013; Todaka

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et al. 2012) but also through post-transcriptional control by mRNA degradation (Belostotsky and Sieburth 2009; Chiba et al. 2013; Floris et al. 2009; Kojima et al. 2011; Pruneda-Paz and Kay 2010; Rayson et al. 2012; Staiger and Green 2011). In addition to the endonucleolytic cleavage process, the majority of mRNA degradation in eukaryotic cells is initiated by poly(A) tail shortening (Belostotsky and Sieburth 2009; Chiba and Green 2009; Meyer et al. 2004). In cytosol, translatable mRNAs interact with poly(A) binding protein (PABP) and eIF4E (Caponigro and Parker 1995), and these mRNP complexes are disrupted after deadenylation; subsequently, either the 5′ cap of the mRNA is removed and the mRNA body is degraded in a 5′–3′ direction by exoribonuclease (XRN) or the 3′ end of the mRNA is attacked by a 3′–5′ exosome complex (Belostotsky and Sieburth 2009; Tharun and Parker 2001; Wilusz et al. 2001). Deadenylation in eukaryotic cell is catalyzed by deadenylases, including PAN2-PAN3 complex, DAN/PARN deadenylase (Korner et al. 1998), and CCR4NOT complex (Chen et al. 2002; Tucker et al. 2001). Despite the functional overlapping of the deadenylases, the CCR4-NOT complex seems to play a major role in the mRNA deadenylation process in most eukaryotes (Parker and Song 2004). The CCR4-NOT complex contains two catalytic subunits, carbon catabolite repressor 4 (CCR4) and CCR4 associated factor 1 (CAF1, also called POP2), both are required for deadenylation. It is believed that CCR4 is the major cytoplasmic deadenylase and acts as the main catalytic component. Evidence from yeast studies shows that point mutation at catalytic residues of CCR4 abolished the CCR4-NOT deadenylase function in vivo; ectopic overexpression of CCR4 complements caf1 defects, whereas ectopic overexpression of CAF1 cannot complement ccr4 defects (Chen et al. 2002; Ohn et al. 2007; Tucker et al. 2001, 2002). However, CAF1 is also essential for deadenylation in yeast, and caf1 mutant strains show defects in deadenylation (Tucker et al. 2001, 2002). In addition, studies on trypanosome and multicellular eukaryotes have indicated that CAF1 also plays a critical role in deadenylation, and controls the expression of certain genes involved in vital physiological processes. Knockdown of CAF1 gene expression resulted in embryonic lethality and delayed larval developmental progress in C. elegans (Molin and Puisieux 2005). In Trypanosoma brucei, deletion of CAF1 delayed deadenylation of bulk mRNAs, and the deadenylase activity of CAF1 was also shown to be essential for cell viability (Schwede et al. 2008). In Drosophila cells, the deletion of CAF1 but not CCR4 delayed the deadenylation of Hsp70 mRNA (Temme et al. 2004). Human cells contain two CCR4 and two CAF1 paralogues that are all involved in deadenylation (Aslam et al. 2009; Funakoshi et al. 2007; Morita et al. 2007; Schwede et al. 2008; Yamashita

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et al. 2005; Zheng et al. 2008). Knockdown of CAF1a and CAF1b or CCR4a and CCR4b resulted in reduced cell proliferation (Aslam et al. 2009; Mittal et al. 2011; Morita et al. 2007). The CCR4-NOT complex also participates in the mechanism involved in RNA-induced gene silencing. When the RNA-induced silencing complex (RISC) is combined with 3′UTR of mRNA, CCR4-NOT complex is recruited to disrupt mRNP structure, reduce translation efficiency and accelerate mRNA degradation of the targeted mRNA by promoting poly(A) removal in mammalian cells (Fabian et al. 2009; Piao et al. 2010). All higher plants, such as Arabidopsis and rice, contain large numbers of CAF1 gene family members (Cai et al. 2011; Walley et al. 2010a, b). In the literature, only a few studies have indicated that the function of CAF1 is cross-related to plant tolerance to abiotic and biotic stress mechanisms. CaCAF1, a pepper CAF1, is required to resist to pathogen infections (Sarowar et al. 2007). Arabidopsis atcaf1a and atcaf1b mutants showed decreased PR1 and PR2 mRNA levels, and the plants were susceptible to pathogen infection (Liang et al. 2009). However, only atcaf1a mutants exhibited phenotypical salt tolerance. Additionally, members of the CAF1 gene, AtCAF1a to AtCAF1k, showed different expression patterns in response to wounding (Walley et al. 2010a). Therefore, it is assumed that functional specificity within CAF1 families is essential for deadenylation of their mRNA targets in response to specific signals. The mRNA degradation process requires the participation of many proteins in deadenylation, decapping, and exoribonucleic cleavage. These proteins are known to colocalize with their target mRNAs in small, discrete, and prominent granular foci known as processing bodies (also termed PBs or P-bodies) within the cytoplasm of eukaryotic cells (Anderson and Kedersha 2006; Eulalio et al. 2007). Deadenylation enzymes including PAN2, PAN3, CCR4, and CAF1 are found to localize in P-bodies (Zheng et al. 2008). Remarkably, P-bodies were undetectable in mammalian cells overexpressing a truncated CAF1 with mutations at catalytic residues; this defect can be restored by coexpression of CCR4 (Zheng et al. 2008), suggesting that deadenylation is a prerequisite for P-body formation and mRNA decay. Arabidopsis Decapping 1 (DCP1), DCP2, VARICOSE (VCS), EXORIBIONUCLEASE 4 (XRN4), PARN, and CCR4a are also colocalized in P-bodies (Moreno et al. 2013; Pomeranz et al. 2010; Weber et al. 2008; Xu and Chua 2009), suggesting that their function has been conserved from yeast to humans and plants through evolution. However, DCP5, identified as an Arabidopsis P-body component and required for P-body formation and postembryonic development, is not associated with VCS (Xu and Chua 2009). Moreover, in contrast to human and yeast eIF4E, Arabidopsis eIF4E homologue protein is localized in plant stress granules but not in

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P-bodies (Weber et al. 2008). These studies have suggested that plant P-bodies contain different components that perform divergent functions. It is still unclear whether the plant CAF1 is localized in P-bodies to perform deadenylation or different plant CAF1 proteins have different subcellular localizations. Rice (Oryza sativa) is one of the world’s most vital crops and is the major staple food of nearly 50 % of the world’s population. However, little is known about rice mRNA deadenylation and there are no reports on the function of rice CAF1. In this study, we cloned and characterized 4 OsCAF1 genes, and showed that the recombinant OsCAF1 proteins exhibited deadenylase activity in vitro. The divergent expression patterns of each member were exhibited in various tissues and were found to response to different abiotic stresses. We provide evidence to indicate that these rice CAF1 proteins are localized in the cytoplasm and nucleus. We further demonstrate that OsCAF1B colocalizes with a P-body marker AtXRN4, indicating that OsCAF1B is a component of the plant P-body.

Materials and methods Identification of CAF1 homologs and phylogenetic analysis To identify the rice and maize CAF1 homologs, we used the AtCAF1a (encoded as At3g44260) sequence (Walley et al. 2010a) as a query to perform the BLAST program, searching the Oryza sativa and Zea mays nodes at Phytozome (www.phytozome.net). Yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), human (Homo sapiens), fruit fly (Drosophila melanogaster), and mouse (Mus musculus) CAF1 amino acid sequences were identified using the NCBI database (www.ncbi.nlm.nih.gov). Finally, all of these amino acid sequences were aligned using the EBI Clustal Ω program (www.ebi.ac.uk), and the phylogenetic trees were generated using MEGA 5 (Tamura et al. 2011). We used the neighbor-joining method to construct different trees with pairwise deletion. The bootstrap calculation was performed using 1,000 replicates for statistical reliability. Plant material, growth conditions, and stress treatment The rice variety used in this study was Oryza sativa L. cv Tainung 67 (TNG67). Seeds were surface-sterilized with 2.5 % NaOCl for 40 min, washed extensively with sterile water, and germinated in the dark on wet filter paper in petri dishes at 37 °C for 48 h. After incubation, uniformly germinated seeds were selected and cultivated in a 500 mL beaker containing half-strength Kimura B solution replaced

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every 3 day. The hydroponically cultivated seedlings were grown in a growth chamber at 28 °C with a 16-h light/8-h dark cycle. Twenty-one-day-old seedlings underwent different stress treatments by replacing the medium. The medium was replaced with a fresh medium prechilled to 4 °C (cold stress), prewarmed to 45 °C (heat stress), supplemented with 200 mM NaCl (salt stress) or 20 μM ABA (ABA treatment). For temperature stress treatments, the seedlings were transferred to growth chambers maintained at 4 °C (cold stress) or 45 °C (heat stress). Drought stress was performed by air-drying seedlings at 28 °C, and samples were taken after 10 and 30 % fresh weight loss was recorded. For wound treatments, the leaves were punctured with needles and then cultured at 28 °C. Samples after the treatments were collected at indicated times, frozen in liquid nitrogen, and stored at −70 °C. Gene expression analyses Total RNA was extracted from the shoots and roots of 21-day-old seedlings treated with ABA or differential stresses by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA gel-blot analysis was performed as described (Ho et al. 2001). Ten micrograms of total RNA was separated on 1 % agarose gel containing 10 mM sodium phosphate buffer (pH 6.5), transferred to a Hybond N1 membrane (GE Healthcare, Pittsburgh, PA, USA) and hybridized at 42 °C with a DNA probe radiolabeled by [α32P] dCTP. For RNA loading controls, 25S, 18S, and 5.8S rDNA in pRY18 (Sano and Sano 1990) were excised with BamHI and used as probes. The quantitative real-time RT-PCR (qRT-PCR) analyses were performed using the FastStart Essential DNA Green Master (Roche, Basel, Switerland) and the iQ5 real-time PCR machine (Bio-RAD, Hercules, CA, USA). Results were repeated independently at least three times and relative gene expression was expressed as a ratio of target gene mRNA to that of rice 18S rRNA. Data were analyzed using the iQ5 2.1 software program provided by the manufacturer. Gene-specific primers used for qRT-PCR are listed in Table S2. Construction of plasmids To construct vectors for the production of recombinant OsCAF1 proteins, full-length OsCAF1s were amplified using PCR by gene-specific primers with cDNA derived from rice suspension cells. The PCR-amplified OsCAF1A, OsCAF1G and OsCAF1H fragments were cloned into pET28b (Merck KGaA, Darmstadt, Germany); OsCAF1B fragment was cloned into pET21a (Merck KGaA). Expression vectors for plant cell subcellular localization (Curtis and Grossniklaus 2003) were constructed using the

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Gateway cloning system as follows. The coding sequences of OsCAF1, AtXRN4, AtfABP2 and AtKSS12 genes without a stop codon were amplified using PCR and subcloned into the pENTR/D-TOPO vector (Invitrogen) to generate entry clones. Using LR clonase (Invitrogen), recombination was conducted to transfer target gene fragments from entry clones to destination vectors, pMDC43, pMDC85 or pMDC139 (Brand et al. 2006; Jia et al. 2007), to generate the N-terminal green fluorescent protein (GFP), C-terminal GFP or C-terminal GUS fusion constructs, respectively. To construct the OsCAF1B-mCherry expression vector, we generated mCherry destination vector by replacing the GFP with mCherry in pMDC43 and pMDC85. Primers used for construction are listed in Table S2. Protein expression and purification Recombinant OsCAF1A, OsCAF1G and OsCAF1H protein expression and purification were performed as previously described (Feddersen et al. 2012) with slight modifications. E. coli BL21 (DE3) containing expression plasmids were grown in 3 L LB medium with kanamycin at 37 °C until a density of OD600 = 0.6–0.8 was reached. Recombinant proteins were induced with 0.5 mM IPTG and incubated at 20 °C overnight. Cells were harvested and resuspended using Buffer A (50 mM Tris–HCl, pH 7.0, 300 mM KCl, 5 mM MgCl2, 5 mM β-mercaptoethanol (BME), 20 mM imidazole, 10 % glycerol and 1 mM PMSF) and lysed by sonication. The crude extract was centrifuged at 12,000 rpm at 4 °C, and then passed through a 0.45-μm nonpyrogenic filter. The protein solution was loaded on a HisTrap column (GE Healthcare) preequilibrated in Buffer A, and then washed with Buffer B (50 mM Tris–HCl, pH 7.0, 1 M KCl, 5 mM MgCl2, and 5 mM BME), and eluted with Buffer C (containing 500 mM imidazole in Buffer A). The elution was dialyzed using buffer D (50 mM Tris–HCl, pH 7.0, 200 mM NaCl, 1 mM MgCl2, 1 mM BME, 10 % glycerol). The recombinant proteins were further purified by gel filtration using HiLoad 26/60 Superdex-75 sizeexclusion column (GE Healthcare) with 50 mM Tris–HCl (pH 7.0), 50 mM NaCl, 1 mM BME, 10 % glycerol. Fraction containing rOsCAF1A, rOsCAF1G or rOsCAF1H was concentrated using a Vivaspin centrifugal concentrator (GE Healthcare), and soluble protein was determined using the Bradford method (Bradford 1976). For OsCAF1B protein expression, E. coli. Rosetta2 (DE3) containing OsCAF1 B expression plasmid was grown in 3 L LB medium with kanamycin at 25 °C until OD600  = 0.6–0.8 was reached. Recombinant proteins were induced with 0.5 mM IPTG and incubated at 20 °C overnight. After sonication, centrifugation and filtration, the total proteins were precipitated by ammonium sulfate (20–40 %). The pellet was dissolved in 50 mM Tris–HCl,

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pH 7.0, 150 mM NaCl and 10 % glycerol, and then desalted by HiPrep desalting FF column (GE Healthcare) and purified by HiPrep Sp FF column (GE heakthcare) and HiLoad 26/60 Superdex-75 size-exclusion column (GE Healthcare). The purified proteins were enriched using centrifugal concentrator and determined by Bradford method. In vitro deadenylase assay The 5′-FAM-labeled RNA substrates, 5′-/56-FAM/UCU AAAUAAAAAAA, were synthesized by Integrated DNA technology (Integrated DNA Technologies, Coralville, Iowa, USA). In vitro enzymatic assay was conducted at 30 °C in 20 μL of a reaction buffer (50 mM Tris–Cl, pH 7.0, 50 mM NaCl, 1 mM MgCl2, 10 % glycerol) with 1 μmol RNA substrate and 1 μg of recombinant OsCAF1 proteins. After incubation, denaturing polyacrylamide gel electrophoresis was performed as previously described (Rio et al. 2010). The RNA gel was visualized using LAS4000 (GE Healthcare). Analysis of subcellular localization The onion bulb epidermis was prepared and particle bombardment was executed as described by (Scott et al. 1999) to introduce GFP, mCherry or GUS expression vectors using a PDS-1000 biolistic device (Bio-Rad) at 1,100 p.s.i. Bombarded specimens were incubated on MS solid medium for 6 h, and were then observed using an Olympus IX71 (Olympus, Tokyo, Japan) inverted fluorescence microscope. Confocal observation was conducted using a Zeiss LSM710 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Serial optical sections of images were captured on a Zeiss LSM710 microscope. For analysis of GUS activity, bombarded onion bulb epidermis were incubated on MS solid medium for 6 h and were then incubated in GUS staining solution at 37 °C overnight, as described by Jefferson et al. (1987). The specimens were observed and photographed using a Zeiss LSM510 microscope (Carl Zeiss Microscopy GmbH). Images were analyzed using Zeiss LSM Image Browser software.

Results Four expressed CAF1 genes were identified from rice To identify rice CAF1 homologs, we used the protein sequence AtCAF1a as a query for the basic local alignment search at Phytozome (www.phytozome.net). Eighteen putative homologs (OsCAF1A-R) were identified from rice genome database (Table S1). However, only four corresponding

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Fig. 1  Amino acid sequence alignments of CAF1 proteins. CAF1 protein sequences of rice (OsCAF1), maize (ZmCAF1), Arabidopsis (AtCAF1), human (HsCNOT7/8), fruit fly (DmCAF1), budding yeast (ScPOP2), and fission yeast (SpCAF1) were aligned using ClustalΩ and edited using GeneDOC. Three conserved DEDD motifs are indicated at the top. Conserved residues, DEDD, are labeled in

red. A fifth residue, histidine, located before the final conserved D, is also highly conserved and labeled in blue. The budding yeast POP2 diverges in the first and final residues indicated in green, and the consensus DEDD is changed to SEDQ. The number of amino acid residues from N-, C-termini or between conserved blocks are indicated in parentheses

cDNA or EST (expressed-sequence tag) sequences of these putative OsCAF1s could be obtained from NCBI and PlantGDB (www.plantgdb.org) database. These four homologs, encoded by LOC_Os08g34170, LOC_Os04g58810, LOC_Os09g24990 and LOC_Os02g55300, were named OsCAF1A, OsCAF1B, OsCAF1G and OsCAF1H, respectively (Table S1). Using the same way to search, we identified 21 AtCAF1a related sequences in maize (Zea mays). Among them, ZmCAF1A.1 (GRMZM2G047019), ZmCAF1A.2 (GRMZM2G123328), ZmCAF1G (GRMZM2G071059), ZmCAF1H.1 (GRMZM2G179633), and ZmCAF1H.2 (GRMZM2G110960) cDNA sequences were available in public databases. The amino acid sequence comparison analysis indicated that the rice and maize CAF1 homologues we identified are well conserved at RNase D domain with three conserved motifs and four important catalytic residues of nuclease activity, DEDD (Fig. 1). In addition, these CAF1 homologues contain the fifth conserved amino acid residue, histidine (Fig. 1). Therefore, all of the identified sequences from rice and maize were grouped in the CAF1 family (ID: PF04857.15). A phylogenetic relationship was constructed using amino acid sequences from rice, maize, Arabidopsis,

human, mouse, fruit fly, and budding and fission yeast CAF1 families. The phylogenetic tree (Fig. 2) showed that all of the Arabidopsis CAF1 proteins could be divided into three groups, consistent with a previous report (Walley et al. 2010a). The four rice CAF1 proteins analyzed in this study were clustered into two groups, I and III. OsCAF1A, OsCAF1G, OsCAF1H were clustered in Group III, whereas OsCAF1B and its counterparts, AtCAF1a and AtCAF1b, were clustered in the same clade, Group I. The amino acid sequences of the OsCAF1 proteins in Group III were more similar to ZmCAF1 than to AtCAF1 proteins. Recombinant OsCAF1 proteins contain deadenylase activity in vitro To determine whether the OsCAF1 proteins contained deadenylation activity, the recombinant His–OsCAF1 proteins were expressed in E. coli. Three recombinant His–OsCAF1 proteins, rOsCAF1A, rOsCAF1G, and rOsCAF1H, were obtained by HisTrap and gel filtration column. Due to a protein aggregation problem of rOsCAF1B protein, we performed ammonium sulfate precipitation, followed by cation-exchange and gel filtration

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448 Fig. 2  Phylogenetic relationships of CAF1 family members. Phylogenetic tree among rice, maize, Arabidopsis, human, mouse, fruit fly, budding and fission yeast CAF1 is generated by MEGA 5 and rooted with SpCAF1 and bootstrap values (>50) are indicated at each node. The members of CAF1 from rice, maize, and Arabidopsis are categorized into Groups, I, II, and III, with at least 50 % bootstrap support. Accession numbers of genes listed here are depicted in the Supplementary Table S2

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to purify intact rOsCAF1B. The deadenylase activities of purified proteins were analyzed by incubating them with 5′-FAM-labeled RNA substrates for various lengths of time. Reaction mixtures were subjected to polyacrylamide gel electrophoresis; the 5′-FAM-labeled fragments were then visualized using fluorometer. rOsCAF1A, rOsCAF1G and rOsCAF1H proteins were able to remove the first seven adenine nucleotides of the substrates within 1 min by stepwise shortening (Figs. 3a, d and e). Compared with rOsCAF1A, rOsCAF1G and rOsCAF1H, the substrates were digested completely by rOsCAF1B within 0.5 min (Fig.  3b), whereas others required longer than 3 min to

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completely digest the substrates (Fig. 3a, d and e). To determine whether the rOsCAF1B exhibited stepwise deadenylase activity as other rOsCAF1 proteins, less amount of rOsCAF1B was examined. As result shown in Fig. 3c, tenfold diluted OsCAF1B proteins (0.1 μg) removed the first seven adenine nucleotides of the substrates by stepwise shortening. These results indicated that the direction of RNA degradation was from 3′ to 5′. The intermediate products (UCUAAAU) accumulated in all of the reaction mixtures. The OsCAF1 proteins removed the uridine nucleotide and the following three adenine nucleotides and produced shorter products FAM/UCU (Fig. 3a–e). To confirm

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Fig. 3  Deadenylase activity assay of recombinant OsCAF1 proteins. Purified rOsCAF1A (a), rOsCAF1B (b), tenfold diluted rOsCAF1B (c), rOsCAF1G (d), rOsCAF1H (e) and a catalytic site mutants, rOsCAF1A(m) (a), rOsCAF1B(m) (b), rOsCAF1G(m) (d) and rOsCAF1H(m) (e) with 5′-FAM labeled synthetic RNA probe

that the deadenylase activity came from the OsCAF1 proteins, a mutation strategy at the conserved active site residues was applied. Four mutated proteins consequently did not exhibit deadenylase activity (Fig. 3a–e), indicating that the deadenylase activity was not from contaminating nucleases during purification. These results indicated that OsCAF1 proteins are active 3′–5′exonucleases.

(f) were used in parallel time-course reactions. The reaction products were subjected to polyacrylamide gel electrophoresis. The FAM images were obtained using a fluorescence imaging system. Commercial RNase A was used as positive control

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Unique and overlapping expression patterns among members of rice CAF1 family To determine the expression profile of the OsCAF1 family in rice, total RNAs were isolated from various tissues and subjected to Northern blot analysis. In the shoots of 21-dayold seedlings and the sheaths of 3-month-old mature plants, OsCAF1A and OsCAF1G mRNA were slightly detectable,

Fig. 4  Expression patterns of OsCAF1 genes in rice. Total RNAs were isolated from roots and shoots of 21-day-old seedlings, and from various tissues of 3-month-old plants. Northern blot analysis was performed using the OsCAF1A-, OsCAF1B-, OsCAF1G- and OsCAF1-specific cDNA regions and 28S rRNA gene as probes. Two bands detected using the OsCAF1B probe are indicated by arrows as OsCAF1B (L) and OsCAF1B (S), respectively. The mRNA of OsCAF1H was not detectable and data are not shown

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and were significantly expressed in the green leaves, senescent leaves, panicle axes, and spikelets of mature plants (Fig.  4a). Two different transcripts of OsCAF1B mRNA, OsCAF1B (L) and OsCAF1B (S), were detected as inverse expression patterns in the various tissues. The OsCAF1B (L) mRNA was detected in roots, sheaths, nodes, and spikelets, whereas OsCAF1B (S) was detected in leaves and senescent leaves (Fig. 4). Northern blot analysis detected no OsCAF1H mRNA in any of the selected tissue (data not shown). To examine whether the expression of each OsCAF1 gene responses to various abiotic stresses, Northern blot analysis and qRT-PCR were used to determine the mRNA levels of OsCAF1 in the roots and shoots of 2-week-old rice seedlings subjected to abscisic acid (ABA) (20 μM), drought (air drying), cold (4 °C), salt (200 mM NaCl), and heat (45 °C) treatments. The accumulation of OsCAF1A mRNA was slightly induced by ABA, salt, cold, and heat both in roots and shoots (Fig. 5a). The level of OsCAF1B (L) mRNA in roots and OsCAF1B (S) mRNA in shoots was significantly induced by drought stress (with 10 or 30 % water loss) and cold stress (Fig. 5a). We further quantified the relative levels of OsCAF1B mRNA, including both forms, in plants subjected to drought and cold stresses. Consistent with the Northern blot analysis, OsCAF1B mRNA levels increased approximately more than 8.5-fold in drought-stressed shoots, when the plants lost more than 10 % of their fresh weight in water (Fig. 5b). The level of OsCAF1B mRNA increased 2.2- and 9.4-fold after shifting to 4 °C for 8 h in roots and shoots, respectively (Fig. 5c). It has been reported that Arabidopsis AtCAF1a and AtCAF1b mRNA accumulated rapidly within 15 min after ABA and wounding treatments (Liang et al. 2009). Since OsCAF1B is closely related to AtCAF1a and AtCAF1b from phylogenic tree, we determined whether the expression of OsCAF1B was in response to ABA and wounding treatments. In seedlings, the accumulation of OsCAF1B mRNA drastically increased within 15 min after wounding and ABA treatments, but then declined 1 h after treatments (Fig. 5d, e). These results indicated that OsCAF1B transcripts were transiently induced by drought, cold, ABA, and wounding treatments. OsCAF1G mRNA was detected in shoots and was slightly repressed by heat stress, ABA and cold stress (Fig.  5a). Moreover, OsCAF1G mRNA was detectable under ABA, drought, salt and heat stress in roots. Among the different stress treatments, OsCAF1H mRNA was only detected under heat stress, and levels of OsCAF1H mRNA increased 9.9-fold in roots and 4.2-fold in shoots after shifting to 45 °C for 1 h, and dramatically decreased to the basal level after 3 h of recovery at room temperature (Fig. 5f). The differential expression patterns of rice CAF1 family members indicate that each OsCAF1 gene in rice may have a unique function in response to various abiotic stresses.

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Differential subcellular localization of OsCAF1 proteins The cellular localization of POP2 (yeast CAF1) and CNOT8 (human CAF1) occurs predominantly in the cytoplasm and P-body (Teixeira and Parker 2007; Tucker et al. 2001; Yamashita et al. 2005; Zheng et al. 2008). Another human CAF1 homolog, CNOT7, is located in the cytoplasm and nucleus (Robin-Lespinasse et al. 2007). Amino acid sequence analysis indicated that some OsCAF1 proteins have various numbers of putative NLS and NES (Fig.  6a). To determine the subcellular localizations of OsCAF1 proteins, OsCAF1 genes were fused with GFP under the control of a 35S promoter and introduced into onion epidermal cells using particle bombardment. To prevent artifacts resulting from high protein expression, GFPemitted fluorescence signals were observed at 6 h after bombardment. As shown in Fig. 6b, fluorescence signals from GFP fused to the C terminal of each OsCAF1 protein (OsCAF1–GFP) were present not only in the nucleus but also in the cytoplasm, the same cellular localization pattern as GFP only. In the cytoplasm, OsCAF1A–, OsCAF1G– and OsCAF1H–GFP protein signals were distributed uniformly (Fig. 6b), whereas OsCAF1B (L)– GFP was localized to specific cytosolic foci and formed filamentous structures (Figs. 6b, c). Similar subcellular localization patterns were observed when GFP fused to the N-terminus of each OsCAF1 (GFP–OsCAF1) (Supplemental Figure 1).We further examined the subcellular localization of OsCAF1 proteins by fusing another reporter, β-glucuronidase (GUS), in onion epidermal cells. Each OsCAF1 fusion proteins (OsCAF1–GUS) were distributed both in the nucleus and cytoplasm, whereas GUS was predominantly localized in the cytoplasm (Supplemental Figure 2). The OsCAF1B–GUS was also localized to specific cytosolic foci and filamentous structures. In contrast to the well-distributed signals of GFP and OsCAF1A–GFP in the nucleus, the OsCAF1B (L)–, OsCAF1G– and OsCAF1H– GFP signals in the nucleus exhibited dense spots that were localized to the nucleoli (Fig. 7). OsCAF1B (L) is colocalized with P‑body marker and tubulin marker Specific localization in the cytosolic foci and filamentous structures of OsCAF1B was observed (Fig. 6b, c). Since CNOT7, a human CAF1 homolog, is known to localize in P-bodies (Zheng et al. 2008), we determined whether OsCAF1B (L) was associated with P-bodies, and conducted colocalization experiments using the Arabidopsis P-body marker EXONUCLEASE 4 (AtXRN4) (Moreno et al. 2013; Souret et al. 2004; Weber et al. 2008). Consistent with Arabidopsis protoplasts, AtXRN4–GFP was localized in cytoplasmic foci resembling P-bodies

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Fig. 5  Expression patterns of OsCAF1 genes under various stress treatments. a Two-week-old rice seedlings subjected to ABA (20  μM), drought (air dry), cold (4 °C), salt (200 mM NaCl), and heat (45 °C) were treated for various periods. Northern blot analysis of total RNAs isolated from rice plants were detected using probes from the OsCAF1A-, OsCAF1B-, OsCAF1G- and OsCAF1H-specific cDNA regions. Ribosomal 25S and 18S RNAs were detected

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with ethidium bromide staining. b–f Total RNAs were isolated from 2-week-old rice seedlings treated by applying drought, cold, ABA, wounding and heat in short periods and subjected to qRT-PCR analysis by using primers specific for OsCAF1B (b–e) and OsCAF1H (f). Relative mRNA expression levels were normalized to the rice 18S rRNA gene

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(A)

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Fig. 6  Subcellular localization of OsCAF1–GFP fusion proteins in onion epidermal cells. a Schematic diagram of rice CAF1 proteins. The gray boxes indicate the CAF1 conserved regions. Putative nuclear localization signals (NLS) and nuclear exclusion signals (NES) of OsCAF1 proteins are indicated as white and black blocks. b The rice OsCAF1A–, OsCAF1B (L)–, OsCAF1G–, and OsCAF1– GFP fusion proteins are localized to the nucleus and cytoplasm. Onion epidermal cells were transformed using constructs, which con-

tain a full-length coding region without a stop codon of the OsCAF1 genes fused to GFP, by particle bombardment. The GFP along is used as the control. c The granules and filamentous structures assembled by OsCAF1B (L)–GFP were detected in the cytoplasm. The green fluorescent signals were obtained using a florescence microscope and are shown in green, and nuclei are indicated by white arrows. Scale bar 50 μm

(Fig.  8a). OsCAF1B (L)–mCherry fluorescent fusion proteins were colocalized with AtXRN4–GFP, indicating that OsCAF1B (L) localized in P-bodies (Fig. 8a). However, certain OsCAF1B (L)–mCherry proteins were not colocalized with AtXRN4–GFP. By contrast, OsCAF1B (L)–GFP proteins assembled as filamentous structures in the cytoplasm are probably microfilaments or microtubules (Fig. 6b, c). We examined whether OsCAF1B (L)–mCherry was associated with a microtubule marker, AtKSS12, Arabidopsis katanin domain one and two (Stoppin-Mellet et al. 2007), or a microfilament marker, AtfABP2, the second actin-binding domain of Arabidopsis fimbrin. The results shown in Fig. 8b indicated that OsCAF1B (L)–mCherry and AtKSS12-GFP were colocalized, wherease AtfABP2-GFP were not (Supplemental Figure 3), indicating that OsCAF1B (L) is associated with microtubules.

Discussion

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The CCR4-NOT complex is known to function in deadenylation, and has been proposed to share a highly conserved mechanism of enzymatic action among eukaryotes. However, the roles of the individual components of the CCR4-NOT complex are not completely consistent among different species. The deadenylation activity of CAF1 is not essential in yeast, whereas it plays a pivotal role in yeast and animal cells (Schwede et al. 2008; Temme et al. 2004; Tucker et al. 2002). In Entamoeba histolytica, the CCR4 homolog gene is not even detectable (Lopez-Rosas et al. 2012). Higher plants contain a number of CAF1 gene family members, unlike animals and yeasts which contain only one or two CAF1 genes. In this study, we identified four homologous rice CAF1 genes and demonstrated that recombinant rice CAF1 proteins exhibited deadenylase

Plant Mol Biol (2014) 85:443–458 Fig. 7  The OsCAF1B (L)–, OsCAF1G– and OsCAF1H– GFP fusion proteins are localized to the nucleolus. Nuclei images of the onion epidermal cells transformed using OsCAF1A-, OsCAF1B (L)– OsCAF1G– and OsCAF1H– GFP constructs. The GFP signals were aggregated to form small granules in nucleoli, observed using a florescence microscope. White arrows indicate nucleoli. Scale bar 50 μm

453

Fluorescence

Bright

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OsCAF1G – GFP

OsCAF1H– GFP

activity. We also analyzed rice CAF1 gene expression and subcellular localization patterns to offer new insights for exploring the functional divergence of the OsCAF1 gene family during rice growth and development, and in response to various stresses. Bioinformatics analysis revealed 8–16 CAF1 homologous genes in the rice genome (Walley et al. 2010b). In this study, we found 18 putative CAF1 genes in rice genome database. However, only OsCAF1A, OsCAF1B, OsCAF1G and OsCAF1H transcripts were identified from rice cDNA or EST sequence database. In addition, no OsCAF1C to OsCAF1F transcripts in rice suspension cultured cells were able to detect by RT-PCR analysis (data not shown). Therefore, it is probable that only OsCAF1A, OsCAF1B,

OsCAF1G, and OsCAF1H are expressed in rice. However, we cannot rule out the possibility that other OsCAF1 genes could be detected under particular conditions. Amino acid sequence alignment and phylogenetic analysis clearly divided these four expressed OsCAF1 proteins into two groups. Regarding classification, OsCAF1A, OsCAF1G, and OsCAF1H are Group III CAF1 proteins. Orthologous pairs of rice and maize CAF1 members are more prevalent than others in the phylogenetic tree, suggesting that ancestor CAF1 genes were shared before the divergence of maize and rice. Within Group III, the subcellular localization patterns of OsCAF1A, OsCAF1G and OsCAF1H were also similar. However, the expression patterns of individual OsCAF1 genes differed in the course of

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AtXRN4-GFP

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mCherry

AtXRN4-GFP

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(B) OsCAF1B -mCherry

AtKSS12 -GFP

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Fig. 8  The OsCAF1B (L) is colocalized to P-bodies and tubulin fibers. a Onion epidermal cells were transformed using OsCAF1B (L)–mCherry and AtXRN4–GFP constructs. The mCherry and GFP images were observed using a florescence microscope. Colocalization of OsCAF1B (L)–mCherry and AtXRN4–GFP were detected in merged images (yellow spots). Some granules indicated by white arrows were not located with AtXRN4 (red spots). The

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mCherry-only construct was used as a control and was not colocalized with AtXRN4. (b) Onion epidermal cells were transformed with OsCAF1B (L)–mCherry and AtKSS12–GFP. The mCherry and GFP images were obtained using a confocal microscope. Fibers containing OsCAF1B (L)–mCherry (red image) were colocalized with a tubulin marker, AtKSS12–GFP (green image). Scale bar 50 μm

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development and in response to various abiotic stresses. For example, OsCAF1A and OsCAF1G were predominately expressed in mature plant green tissues; OsCAF1G mRNA was not detectable in the roots of seedlings; OsCAF1H was induced only by heat stress. Thus, although functional redundancies among OsCAF1s are extensive, individual OsCAF1 genes may play specific roles during biological processes such as sub- or neofunctionalization of individual CAF1 protein in plants as described by Walley et al. (2010a, b). By contrast, OsCAF1B was clustered in Group I with the biotic and abiotic stress-related proteins, AtCAF1a and AtCAF1b. Moreover, the transcript levels of OsCAF1B peaked at 15 min and decreased rapidly in response to wounding, consistent with AtCAF1a and AtCAF1b (Liang et al. 2009). This suggests that the mechanism regulating the expression of the response to wounds exhibited by OsCAF1B, AtCAF1a, and AtCAF1b was conserved and derived from the same ancestor. The two transcript forms of OsCAF1B, OsCAF1B (L) and OsCAF1B (S), were observed in Northern blot analysis, and their tissue expression patterns did not overlap but were both induced by cold, drought, and ABA. Furthermore, genomic Southern blot analysis using the OsCAF1B coding region as a probe was able to detect only one hybridization signal (Supplementary Figure 4). Therefore, we assume that these two OsCAF1B transcripts are derived from the same gene LOC_Os04g58810. Two forms of OsCAF1B cDNA clones, 006-203-G11 (OsCAF1B (L)) and J023025J17 (OsCAF1B (L)) are reported in public available database. The deduced amino acid sequence of putative OsCAF1B (S) cDNA contains the conserved RNase D motif, but not the second NLS (Supplementary Figure 5). However, we were not able to clone OsCAF1B (S) cDNA successfully from neither leaves, calli nor suspension cultured cells of rice. Future studies to determine whether OsCAF1B encodes two isoforms of OsCAF1B proteins might provide more information on the specificity of the OsCAF1B function in rice. In our subcellular localization analysis, fluorescent reporter proteins fused to OsCAF1A, OsCAF1G, or OsCAF1H did not form any granule-like structures within the cytoplasm. The uniform distribution of these three OsCAF1 proteins in the cytoplasm is consistent with yeast CAF1 proteins that do not form punctate structures under normal conditions. However, yeast CAF1 can be recruited to P-bodies under glucose-deprived or stress condition (Teixeira and Parker 2007). Considering the differential expression patterns of each rice CAF1 member, we propose that OsCAF1A, OsCAF1G, and OsCAF1H may relocalize to P-bodies under specific conditions; for example, OsCAF1H under heat stress. However, OsCAF1B (L) was clearly localized in P-bodies, similar to CAF1 in human and Drosophila cells (Temme et al. 2010; Zheng et al.

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2008). Although it is still unclear whether the localization of CAF1 in P-bodies is important for mRNA deadenylation, this study showed that one rice CAF1 member was localized in P-bodies and may play a certain role there. We found that more cytosolic foci were formed by OsCAF1B (L)–mCherry than AtXRN4-GFP, indicating that OsCAF1B could be a component of granules other than P-bodies. Arabidopsis CCR4a is known to locate both in P-bodies and siRNA-bodies (Moreno et al. 2013). Additionally, XRN1, eIF4E, tristetraprolin (TTP), and others have been shown to be components of P-bodies and stress granules (Kedersha et al. 2005). In addition to the granule structure, filamentous structures in the cytoplasm are assembled by OsCAF1B (L)–GFP and OsCAF1B (L)–mCherry. The OsCAF1B (L) filamentous structures were colocalized with microtubules. Recently, P-bodies and stress granules have been reported for their association with microtubules (Aizer et al. 2008; Loschi et al. 2009; Sweet et al. 2007). Further study will be required to address whether OsCAF1B (L) locates to stress granules and siRNA-bodies; such study will provide insights into the dynamic function of rice CAF1 members. The CCR4-NOT complex has been shown to participate in many processes in the nucleus, including transcription, DNA repair, histone modification, chromatin remodeling, nuclear RNA quality control, and mRNA export (Collart and Panasenko 2012; Jayne et al. 2006; Mulder et al. 2005, 2007; Zwartjes et al. 2004). However, most components of CCR4-NOT are detected predominantly in the cytoplasm in yeasts, mammals, fruit flies, and trypanosomes, in addition to the nucleus (Lau et al. 2010; Schwede et al. 2008, 2009; Temme et al. 2010; Tucker et al. 2001; Yamashita et al. 2005). OsCAF1–GFP fusion proteins may diffuse passively into the nucleus, but functions of OsCAF1 proteins in the nucleus are worthy to study. In addition, OsCAF1B (L), OsCAF1G, and OsCAF1H (except to OsCAF1A) were detected in the nucleus with dense spots localized in the nucleolus, implying that rice CAF1 proteins may participate known biogenesis and processing of rRNA in the nucleolus. Alternatively, CAF1 might participate in RNA quality control, a process closely related to the nucleolus and poly(A) tails, including snRNA, tRNA, rRNA, and pre-mRNA. A previous study reported that a physical interaction and functional connection exists between CCR4-NOT and the TRAMP complex (Azzouz et al. 2009), which is involved in the quality control of snoRNAs, snRNA, tRNA, rRNA and pre-mRNA (Azzouz et al. 2009; Dez et al. 2006; Doma and Parker 2007; Egecioglu et al. 2006; Fang et al. 2004; Kadaba et al. 2004; LaCava et al. 2005; Wyers et al. 2005). Mutations in CAF1 and other components of the CCR4-NOT complex could potentially cause the accumulation of polyadenylated snoRNAs (Azzouz et al. 2009). Future studies will be

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necessary to determine whether this model can support rice CAF1 action in the nucleolus. CCR4-NOT complex contains several subunits that have been demonstrated to play critical roles for CCR4-NOT complex in yeast and mammalian cells. Currently, the role of CCR4, a deadenylase component of CCR4-NOT complex, is not well investigated in plants. There are several putative genes of CCR4 homologs in rice and Arabidopsis (Dupressoir et al. 2001; Winkler and Balacco 2013). However, their deduced amino acid sequences are less conserved in N-terminal region of CCR4 in yeast and mammalian cells. For example, plant CCR4 homologs lack the leucine-rich repeat motif that is required for interaction with CAF1 proteins in yeast and mammalian cells (Clark et al. 2004; Mittal et al. 2011). In present study, we characterized 4 CAF1 homologs in rice. If rice CCR4 does interact with CAF1, another architecture may exist between CCR4 and CAF1 in rice. Acknowledgments  We thank Dr. Su-May Yu and Ms. Sue-Ping Lee at the Institute of Molecular Biology, Academia Sinica, Taipei, for technical assistance in confocal microscopy. This work was supported by a Grant (100-2321-B-008-003-MY3) from the National Science Council of the Republic of China.

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