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Arabidopsis Deadenylases AtCAF1a and AtCAF1b PlayOverlapping and Distinct Roles in MediatingEnvironmental Stress Responses1[C][W][OA]

Justin W. Walley, Dior R. Kelley, Gergana Nestorova2, David L. Hirschberg3, and Katayoon Dehesh*

Department of Plant Biology (J.W.W., K.D.) and Department of Molecular and Cellular Biology (D.R.K.),University of California, Davis, California 95616; and Stanford School of Medicine, Stanford University,Stanford, California 94305 (G.N., D.L.H.)

To maintain homeostasis in an ever-changing environment organisms have evolved mechanisms to reprogram geneexpression. One central mechanism regulating gene expression is messenger RNA (mRNA) degradation, which is initiatedby poly(A) tail shortening (deadenylation). The carbon catabolite repressor 4-CCR4 associated factor1 (CCR4-CAF1) complex isthe major enzyme complex that catalyzes mRNA deadenylation and is conserved among eukaryotes. However, thecomponents and functions of this global regulatory complex have not been well characterized in plants. Here we investigatethe CAF1 family in Arabidopsis (Arabidopsis thaliana). We identify 11 AtCAF1 homologs and show that a subset of these genesare responsive to mechanical wounding, among them are AtCAF1a and AtCAF1b whose expression levels are rapidly andtransiently induced by wounding. The differential expression profiles of the various AtCAF1s suggest that not all AtCAF1genes are involved in stress-responsive regulation of transcript levels. Comparison of misexpressed genes identified viatranscript profiling of Atcaf1a and Atcaf1b mutants at different time points before and after wounding suggests that AtCAF1aand AtCAF1b target shared and unique transcripts for deadenylation with temporal specificity. Consistent with the AtPI4Kg3transcript exhibiting the largest increase in abundance in Atcaf1b, AtCAF1b targets AtPI4Kg3 mRNA for deadenylation. Stress-tolerance assays demonstrate that AtCAF1a and AtCAF1b are involved in mediating abiotic stress responses. However,AtCAF1a and AtCAF1b are not functionally redundant in all cases, nor are they essential for all environmental stresses. Thesefindings demonstrate that these closely related proteins exhibit overlapping and distinct roles with respect to mRNAdeadenylation and mediation of stress responses.

Communication of developmental and environmen-tal information is mediated through alteration of geneexpression. The steady-state level of messenger RNA(mRNA) within a cell is determined by the combina-tion of the rate of transcription and the rate of post-transcriptional processes regulating mRNA decay.Genome-wide approaches are revealing that in re-sponse to environmental stimuli there is a large

reprogramming of gene expression (Eulgem, 2005;Swindell, 2006; Kilian et al., 2007; Ma and Bohnert,2007; Walley et al., 2007; Walther et al., 2007; Shalemet al., 2008). While changes in the rate of mRNAdegradation provide a rapid mechanism to altermRNA abundance, research has largely focused onchanges in transcriptional regulation as a means tocontrol mRNA levels (Gutierrez et al., 2002; Yamashitaet al., 2005; Narsai et al., 2007; Belostotsky and Sieburth,2009; Chiba and Green, 2009; Lee and Glaunsinger,2009).

In eukaryotes, degradation of mRNA begins withshortening of the poly(A) tail, referred to as dead-enylation, which is the rate-limiting step (Meyer et al.,2004; Belostotsky and Sieburth, 2009; Chiba and Green,2009). The process of deadenylation has been beststudied in yeast (Saccharomyces cerevisiae) where it hasbeen shown that the carbon catabolite repressor4-CCR4 associated factor1 (CCR4-CAF1; also calledPop2p) complex serves as the major deadenylasecomplex (Tucker et al., 2001). In addition to theCCR4-CAF1 complex, both the poly(A) ribonuclease(PARN), for which mutants in Arabidopsis (Arabidop-sis thaliana) are embryo lethal, and poly(A) nucleaseare also active deadenylases (Chiba et al., 2004; Meyeret al., 2004; Reverdatto et al., 2004; Belostotsky andSieburth, 2009). Following deadenylation, mRNA de-

1 This work was supported by the National Science Foundation(grant nos. 0543904 and 0606838 to K.D.) and a National Institutes ofHealth training grant in cellular and molecular biology (grant no.T32 GM070377 to J.W.W.).

2 Present address: Biomedical Engineering, Louisiana Tech Uni-versity, Ruston, LA 71270.

3 Present address: Clinical Pathology, Columbia University, NewYork, NY 10032.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Katayoon Dehesh ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.109.149005

866 Plant Physiology�, February 2010, Vol. 152, pp. 866–875, www.plantphysiol.org � 2009 American Society of Plant Biologists

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cay proceeds via two distinct pathways. In one path-way mRNA is degraded in a 3#-to-5# manner via theexosome (Chekanova et al., 2007; Belostotsky andSieburth, 2009). Alternatively, deadenylated mRNAcan also undergo 5#-to-3# decay, which first requiresthat the 5# cap is removed from the deadenylatedmRNA (Goeres et al., 2007; Belostotsky and Sieburth,2009).The biological role of CAF1 has been examined in

a range of eukaryotic species. In yeast, caf1 deletionstrains are sensitive to high temperatures and caf-feine (Hata et al., 1998). Analysis of CAF1 inCaenorhabditis elegans using RNAi and deletion al-leles demonstrates that CAF1 is essential for embry-onic and larval development (Molin and Puisieux,2005). Additionally, CAF1 is required for normalgrowth of trypanosomes (Schwede et al., 2008).Finally, mice lacking CAF1 are sterile (Nakamuraet al., 2004).In plants, limited experiments have investigated

components of the CCR4-CAF1 complex. However,the studies that have been conducted focus on CAF1and demonstrate a role for CAF1 in both develop-ment as well as response to biotic stresses. InArabidopsis, AtCAF1 homologs have been shownto be induced by hormone treatment as well asabiotic and biotic stresses (Lee et al., 2005; Zhenget al., 2006; Ferrari et al., 2007; Walley et al., 2007;Liang et al., 2009). Additionally, one study foundthat overexpression of a yeast CAF1 homolog frompepper (Capsicum annuum; CaCAF1) in tomato (Sola-num lycopersicum) resulted in growth enhancementand resistance to the oomycete pathogen Phytoph-thora infestans. Conversely, when CaCAF1 is silencedin pepper there is significant growth retardation andplants are susceptible to the pathogen Xanthomonasaxonopodis pv vesicatoria (Sujon et al., 2007). Further-more, in Arabidopsis it has been demonstrated thattwo yeast CAF1 homologs, AtCAF1a (At3g44260)and AtCAF1b (At5g22250), are active deadenylasesin vitro and partially complement growth defects ofa yeast caf1 deletion strain. Finally, Arabidopsismutant lines Atcaf1a-1 and Atcaf1b-1 are susceptibleto Pseudomonas syringae pv tomato DC3000 (Lianget al., 2009).In this study we examine the role of the Arabidop-

sis CAF1 family of deadenylases in abiotic stressresponses. We identify a total of 11 AtCAF1 homologsthat exhibit differential patterns of expression inresponse to wounding. Transcriptional profiling ofAtcafa-1 and Atcaf1b-2 suggests that AtCAF1a andAtCAF1b target unique transcripts for deadenylationwith temporal specificity. Additionally, we show thatAtCAF1b targets a putative phosphatidylinositol4-kinase mRNA for deadenylation. Finally, stress-tolerance assays suggest that while AtCAF1a andAtCAF1b are involved in mediating responses to anumber of environmental stresses, they do not actredundantly in all cases nor are they required for allstresses.

RESULTS

Identification of the CAF1 Family in Arabidopsis

We previously identified two Arabidopsis homologsof the yeast CAF1 protein, AtCAF1a and AtCAF1b, viatranscript profiling, as rapid wound response (RWR)genes (Walley et al., 2007). To determine the fullcomplement of AtCAF1 homologs present in theArabidopsis genome, we used the AtCAF1a sequenceto identify all proteins with sequence similarity using abasic local alignment search tool (BLAST; Altschulet al., 1990) at Phytozome (www.phytozome.net). Thissearch yielded nine additional sequences, which wenamed AtCAF1c to AtCAF1k (Figs. 1 and 2), resultingin a small family of 11 members. Alignment theAtCAF1 proteins with CAF1 sequence from othereukaryotes demonstrates that the RNaseD domain(Daugeron et al., 2001; Sujon et al., 2007) is wellconserved in the AtCAF1 family (Fig. 1). We alsofound two closely related sequences in moss (Physco-mitrella patens), eight sequences in rice (Oryza sativa),and 10 sequences in poplar (Populus trichocarpa; datanot shown), demonstrating that there has been asignificant expansion of the CAF1 gene family inangiosperms, possibly due to gene duplication events.In other eukaryotes such as yeast, fly, mouse, andhuman there are only one or two CAF1 genes (Fig. 2;Albert et al., 2000; Daugeron et al., 2001; Nakamuraet al., 2004; Temme et al., 2004; Yamashita et al., 2005).

To determine which of the AtCAF1 sequences aremost closely related to the yeast CAF1 protein we firstperformed an alignment of all 11 AtCAF1 proteinsequences with the yeast, fly, mouse, and human CAF1sequences using Se-Al and CLUSTALW. The align-ment was trimmed to include only conserved regions(data not shown). Using this alignment we ran aparsimony analysis in PAUP* (Harrison and Langdale,2006). We obtained 10 trees from this analysis, whichwere then computed into a consensus tree using thestrict option in PAUP* (Fig. 2). The tree was thenrooted with Saccharomyces pombe CAF1 and bootstrapvalues were calculated in PAUP*. The obtained con-sensus tree places all of the AtCAF1s as sister to yeastCAF1, showing that while AtCAF1a to AtCAF1k arerelated to the yeast protein orthology is equivocal (Fig.2). The Arabidopsis homologs group into three well-supported clades: AtCAF1c to AtCAF1g; AtCAF1h toAtCAF1k; and AtCAF1a and AtCAF1b (Fig. 2). Thesephylogenetic relationships suggest that there could befunctional overlap between clade members. Given thelarge number of AtCAF1 homologs it is also possiblethat either sub- or neofunctionalization has occurredamong some of the family members.

The AtCAF1 Gene Family Exhibits DifferentialExpression Patterns in Response to Stress

It has previously been shown that AtCAF1a andAtCAF1b respond rapidly and transiently to a rangeof stimuli including mechanical wounding, methyl

Messenger RNA Decay Mediates Stress Responses

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jasmonate, abscisic acid, 1-aminocyclopropane-1-carboxylic acid, salicylic acid, and Pseudomonas syringaepv tomatoDC3000 (Walley et al., 2007; Liang et al., 2009).To determine if stress-induced transcriptional activa-tion of AtCAF1a and AtCAF1bwas unique or a generalproperty of all 11 family members, we examined theexpression of AtCAF1a to AtCAF1k in response tomechanical wounding by quantitative reverse tran-scription (qRT)-PCR. These data show that five of the11 AtCAF1 genes are up-regulated in response to

mechanical stress, while four of the 11 AtCAF1 genesdo not respond to mechanical wounding and one isdown-regulated (Fig. 3). Finally, we could not detectexpression of AtCAF1f under the conditions we exam-ined. The differential expression patterns exhibited bythe AtCAF1 family members suggests that a subset ofthe AtCAF1 genes may be involved in rapid stress-responsive regulation of transcript levels while othersmay regulate deadenylation of mRNAs responsive tovarious developmental and environmental cues.

Figure 1. ClustalW generated alignment of CAF1 protein sequences from Arabidopsis, pepper (CaCAF1), yeast (SpCAF1), human(HsCNOT7/8), and Drosophilia (DmPOP2). Conserved RNase D-domain residues are shaded in color. [See online article forcolor version of this figure.]

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Transcript Profiling of AtCAF1a and AtCAF1b Mutants

For further investigation into the role of the Arabi-dopsis CAF1 homologs in regulating gene expressionand stress tolerance we focused on AtCAF1a andAtCAF1b. These two AtCAF1 family members wereselected because (1) they were identified from previ-ous studies as RWR genes (Walley et al., 2007) and (2)they form a monophyletic clade, suggesting that theymay exhibit functional overlap. Many of the RWRgenes are also categorized as abiotic and biotic stress-responsive genes, suggesting that they likely comprisea set of core components in the general stress-responsenetwork (Walley et al., 2007). We were therefore inter-

ested in elucidating what role(s) AtCAF1a and AtCAF1bmay play during signaling upon environmental stress.Towards this aim we obtained Salk T-DNA insertionlines for AtCAF1a and AtCAF1b and confirmed thatthey do not produce full-length transcripts and arelikely functional nulls (Supplemental Fig. S1). Thesemutant lines are visually indistinguishable form thewild-type plants.

We hypothesized that AtCAF1a and AtCAF1b couldinteract with target mRNAs to result in altered geneexpression profiles. To test this hypothesis we per-formed transcript profiling using the Agilent V3 oligomicroarray, which contains probes for 28,500 genesfrom The Institute for Genomic Research’s ATH1 v.5annotation as well 10,000 probes based on MassivelyParallel Signature Sequencing data (Meyers et al.,2004) and identified genes that are misexpressed inthe absence of AtCAF1a and AtCAF1b. For this exper-iment we examined 3-week-old rosette leaves fromwild-type, Atcaf1a-1, and Atcaf1b-2 plants, both beforeand intervals after wounding, using three indepen-dent biological replicates for each genotype and timepoint. The full set of the raw intensity microarray dataare deposited at http://www.ncbi.nlm.nih.gov/geo/,under Gene Expression Omnibus (GEO) accessionGSE17022. Misexpressed genes were classified asprobes that exhibited an increase or decrease in signalof at least 2-fold and had a P value # 0.05. Using thisapproach, we found that between 62 and 136 geneswere misexpressed in Atcaf1a-1 compared to wildtype, depending on the time point (Fig. 4; Supplemen-tal Table S1). While a smaller number of genes (12–28)were misexpressed in Atcaf1b-2 compared to wild type(Fig. 4; Supplemental Table S1). The number of up- anddown-regulated genes in each of the cafmutant lines atvarious intervals post wounding are shown (Supple-

Figure 2. Phylogenetic tree of the CAF1 family from Arabidopsis.Related sequences from yeast (S. cerevisiae and S. pombe), human,mouse, and fly are also included. While most eukaryotes contain onlyone or two CAF1 proteins, Arabidopsis has 11, which group into threewell-supported clades. Clade I includes AtCAF1a and AtCAF1b; clade IIincludes AtCAF1c to AtCAF1g; and clade III includes AtCAF1j andAtCAF1k. Bootstrap values on nodes were generated in PAUP*.

Figure 3. Expression of the AtCAF1 gene familyin response to wounding. Total RNA was ex-tracted from 3-week-old rosette leaves before andat intervals after mechanical wounding and sub-jected to qRT-PCR analysis. Transcript levels werenormalized to internal control genes (At4g34270and At4g26410) measured in the same samples.Data are means of three independent biologicalreplicates 6SEM.



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mental Table S2).Atcaf1a-1 andAtcaf1b-2misexpressedgenes were classified according to gene ontology (GO)terms to provide insight into their biological function(Berardini et al., 2004). The two largest defined classesof GO terms involve response to stress or abiotic/biotic stimuli (Supplemental Fig. S2), suggesting thatthese two AtCAF1 genes are involved in stress re-sponses. Comparison of the misexpressed genes re-vealed a limited temporal overlap for both Atcaf1a-1 aswell as Atcaf1b-2 (Fig. 4A). Additionally, there is lim-ited overlap in transcripts that are misexpressed in theabsence of either AtCAF1a or AtCAF1b at 0, 30, or 90min post wounding (Fig. 4B). While there is minimaloverlap in the transcript profiles, the overlap that isobserved is greater than would be expected by ran-dom chance (Fig. 4). The microarray results were nextvalidated by qRT-PCR for five selected misexpressedgenes (Figs. 5 and 6; Supplemental Fig. S3). Theexpression levels determined by qRT-PCR data are ingood agreement with those observed by transcriptprofiling with a spearman rank order correlation co-efficient of 0.938 (P value = 0.000; Sokal and Rohlf,

1995). These results collectively indicate that AtCAF1aand AtCAF1b target shared and unique transcripts fordeadenylation with temporal specificity.

AtCAF1b Is Required for Deadenylation ofAtPI4Kg3 mRNA

We hypothesized that direct mRNA targets ofAtCAF1s would not be deadenylated in caf1 mutantsand would therefore show an increase in abundance inthe absence of AtCAF1a or AtCAF1b. The gene thatexhibited the greatest increase in abundance observedfor either of the Atcaf1 mutants encodes a putativephosphatidylinositol 4-kinase (AtPI4Kg3; Mueller-Roeber and Pical, 2002). In Atcaf1b-2 transcript levelsof AtPI4Kg3 were increased 42- to 63-fold in ourmicroarray, suggesting that it may be a direct targetof AtCAF1b (Supplemental Fig. S3). We confirmed by

Figure 4. Comparison of Atcaf1a and Atcaf1b dependent transcriptprofiles. A, Overlap between genes misexpressed at 0, 30, and 90 minpost wounding in either Atcaf1a-1 or Atcaf1b-2. B, Comparison ofAtcaf1a-1 versus Atcaf1b-2 misregulated genes at each time point. Theminimal overlap observed for all comparisons is statistically significantat a P value of at least 3.043 1028. Misexpressed genes in each mutantwere determined based on an increase or decrease in expression levelof at least 2-fold (P# 0.05) relative to wild type at each respective timepoint.

Figure 5. Validation of Atcaf1a and Atcaf1b misexpressed genes.Normalized expression values from microarray (left) and qRT-PCR(right) for selected genes. Data are means of three independentbiological replicates 6SEM. For qRT-PCR, transcript levels were nor-malized to internal control genes (At4g34270 and At4g26410) mea-sured in the same samples. WT, Wild type.

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qRT-PCR that AtPI4Kg3 levels were indeed increasedexclusively in Atcaf1b-2 (Fig. 6A). To test if AtCAF1b isacting directly onAtPI4Kg3mRNAwe performed poly(A) tail length (PAT) assays (Salles and Strickland,1999). As shown in Figure 6B the length of AtPI4Kg3mRNA increased specifically in Atcaf1b-2. Addition-ally, the increase in length of AtPI4Kg3 mRNA inAtcaf1b-2 is not a result of a general increase in PAT, asother transcripts tested, including UBQ10, did notincrease in size (Fig. 6C; data not shown). Takentogether these results strongly suggest that AtCAF1btargets AtPI4Kg3 mRNA for deadenylation. We alsotested, via the PAT assay, a number of transcripts thatwere increased in abundance in Atcaf1-a, albeit to amuch lower level than AtPI4Kg3 was in Atcaf1b-2, andwere unable to uncover any potential targets of CAF1a(data not shown), likely due to the low sensitivity ofthe PAT assay.

AtCAF1a and AtCAF1b Regulate AbioticStress Responses

AtCAF1a and AtCAF1b were originally identified asRWR genes, which are enriched for abiotic and bioticstress-responsive genes, suggesting that these CAF1smay be involved in mediating multistress resistance.Additionally, it was recently shown that both AtCAF1aand AtCAF1b are required for resistance to the path-ogen Pseudomonas syringae (Liang et al., 2009). Wetherefore chose to test both Atcaf1a and Atcaf1b mu-tants for tolerance to various environmental stresses.We first assayed survival rate of seedlings germinatedon the reactive oxygen species inducer methyl viol-ogen (MV; paraquat). This experiment demonstratedthat both Atcaf1a and Atcaf1b are more sensitive to MVthan wild type (Fig. 7A; Supplemental Fig. S4). We alsotested their ability to cope with salt stress by germi-nating seeds on salt plates. Atcaf1a plants show an

increased germination rate on 200 mM NaCl comparedto wild type and Atcaf1b (Fig. 7, B and C). No differ-ences in survival or germination were observed forplants grown on control plates (Fig. 7C; data notshown). However, Atcaf1a and Atcaf1b mutants didnot differ in tolerance to waterlogging or the pathogenBotrytis cinerea when compared to wild type (Fig. 7, Dand E). These data suggest that while AtCAF1a andAtCAF1b are involved in mediating response to bothabiotic and biotic stresses they do not act redundantlyin all cases nor are they required for all stresses.

DISCUSSION

In eukaryotes control of mRNA degradation is anessential component in the regulation of transcriptabundance (Meyer et al., 2004; Belostotsky and Sieburth,2009; Chiba and Green, 2009). As deadenylation is therate-limiting step in mRNA decay it is critical formaintaining appropriate levels of mRNA. The CCR4-CAF1 complex forms the major deadenylation com-plex in yeast, where it has been extensively studied(Tucker et al., 2001; Chiba and Green, 2009). In eukary-otes, such as yeast, fly, mouse, and human, CAF1homologs have been identified and shown to be im-portant for deadenylation. Furthermore, in these spe-cies there are only one or two CAF1 genes (Albert et al.,2000; Daugeron et al., 2001; Nakamura et al., 2004;Temme et al., 2004; Yamashita et al., 2005). In contrast,our results demonstrate that there has been an expan-sion of the CAF1 gene family in angiosperms (Fig. 2;data not shown). Given the large number of AtCAF1homologs in Arabidopsis it is possible that sub- orneofunctionalization has occurred among some of thefamily members, resulting in distinct CAF1 proteinsfunctioning under specific conditions and/or target-ing unique transcripts. In support of this idea qRT-

Figure 6. AtPI4Kg3 PAT is stabilized in Atcaf1b. A,qRT-PCR of AtPI4Kg3 in response to wounding,normalized to internal control genes (At4g34270and At4g26410) measured in the same samples(n = 36 SEM). WT, Wild type. B, PATassay measuringthe PAT of AtPI4Kg3 in wild type, Atcaf1a-1, andAtcaf1b-2 before and in response to mechanicalwounding. C, PAT assay measuring the PAT ofUBQ10 in wild type, Atcaf1a-1, and Atcaf1b-2 be-fore and in response to mechanical wounding.



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PCR analysis of the AtCAF1 family revealed thatAtCAF1 genes exhibit differential expression patternsin response to mechanical wounding (Fig. 3). Thisdifferential expression behavior exhibited by AtCAF1family members is consistent with the processes ofsub- and/or neofunctionalization.

In a recent review Belostotsky and Sieburth (2009)hypothesized that deadenylation complexes in plantsmay act nonredundantly and target specific subsets oftranscripts. This hypothesis is based on the findingthat Arabidopsis parn (a second type of deadenylase)mutants are embryo lethal and that only a subset ofembryo-expressed genes have increased PAT in parnmutants (Reverdatto et al., 2004). In support of thishypothesis our results suggest that AtCAF1a andAtCAF1b are also able to target specific transcripts.First, the transcript-profiling experiment reveals thatloss of AtCAF1a or AtCAF1b leads to largely distinctsets of altered transcripts. Additionally, there is limitedoverlap between the three time points assayed for bothAtcaf1a as well as Atcaf1b (Fig. 4), suggesting that thesedeadenylases act with temporal specificity. Second, theexpression of AtPI4Kg3 is increased specifically in

Atcaf1b. The increased expression of AtPI4Kg3 is con-sistent with the observed increase in PAT specificallyin the absence of AtCAF1b, further demonstrating thatAtCAF1a and AtCAF1b are capable of targeting spe-cific transcripts (Fig. 6). Lastly, while Atcaf1a andAtcaf1b exhibit similar responses to most environmen-tal stresses, Atcaf1a is more tolerant to salt treatment.These results demonstrate a functional specificity be-tween these two paralogs, which would be consistentwith a sub (or neo) functionalization event (Fig. 7).Taken together these expression and stress analysesshow that not only do the different types of dead-enylation (PARN versus CCR4-CAF1) complexes tar-get unique transcripts but also that closely relateddeadenylases are able to target unique mRNAs, re-sulting in unique biological responses.

The ability of AtCAF1a and AtCAF1b to targetspecific mRNAs raises the question of how this spec-ificity is achieved. In Drosophila, the interaction ofCAF1 with the RNA-binding protein Smaug allowsfor the specific targeting of the CCR4-CAF1 complexto Hsp83 and nanos mRNAs (Semotok et al., 2005;Zaessinger et al., 2006). Additionally, in yeast the

Figure 7. AtCAF1a and AtCAF1b mediate re-sponse to abiotic stresses. A, Survival of wildtype (WT), Atcaf1a-1, and Atcaf1b-2 on 5 mM MV(paraquat). Data are means of nine independentbiological replicates 6SEM. Asterisks denote asignificant difference of both Atcaf1a and Atcaf1bfrom wild type (P, 0.05) as determined by t tests.B, Germination of wild type, Atcaf1a-1, andAtcaf1b-2 on 200 mM NaCl. Data are means offour independent biological replicates 6SEM. As-terisks denote a significant difference of Atcaf1afrom wild type and Atcaf1b (P , 0.05) as deter-mined by t tests. C, Wild type, Atcaf1a-2, andAtcaf1b-3 grown on either 0 mM NaCl for 2 d or200 mM NaCl for 12 d. D, Waterlogging toleranceof wild type, Atcaf1a-1, and Atcaf1b-2. Three-week-old plants were submerged for 7 d and thenallowed to recover for 5 d prior to measurementof fresh weight. Data are means of 32 indepen-dent biological replicates6SEM. E, Lesion area 4 dafter spot inoculation with spores of B. cinereaisolate B05.10. Similar results were obtainedusing the B. cinerea isolate DN (data not shown).Data are means of 15 independent biologicalreplicates 6SEM. All abiotic and biotic stress-tolerance assays were independently repeatedone to four times with similar results.

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RNA-binding protein Mpt5p directly interacts withCAF1 and recruits the CCR4-CAF1 complex specifi-cally toHOmRNA (Goldstrohm et al., 2006). Given thehigh degree of conservation among eukaryotic CAF1homologs we hypothesize that AtCAF1 mRNA targetsmay be identified in a similar fashion via associationwith specific RNA-binding proteins. These character-istic CAF1-RNA-binding protein complexes wouldthen enable deadenylation of distinct mRNA sub-strates.The limited studies investigating CAF1 function in

plants have focused on the role of these genes inmediating biotic stress responses. Initially, it wasreported that a pepper CAF1 homolog, CaCAF1, isrequired for resistance to the pathogen Xanthomonasaxonopodis pv vesicatoria (Sujon et al., 2007). Addition-ally, Liang et al. (2009) reported recently that AtCAF1aand AtCAF1b act redundantly in defense against thepathogen P. syringae pv tomato DC3000, which corre-lates with reduced levels on PR1 expression in non-treated Atcaf1 mutant plants. Consistently, we alsoassayed the steady-state levels of PR1 via qRT-PCRand found reduced levels in both Atcaf1a-1 andAtcaf1b-2 plants (data not shown). The results of ourstress assays add a number of important observationstoward the understanding of the biological role CAF1proteins play in plants. First, we found that AtCAF1aand AtCAF1b do not act redundantly, in all cases, inresponse to stress (Fig. 7, B and C; Supplemental Fig.S4). In addition, AtCAF1s are not simply required forstress tolerance but in certain cases they can actuallyact as a negative regulator of stress tolerance (Fig. 7, Band C). We also show that AtCAF1a and AtCAF1bplay a role in abiotic stress responses (Fig. 7, A–C;Supplemental Fig. S4). Finally, while AtCAF1a andAtCAF1b mediate resistance toward both abiotic andbiotic stresses, they are not involved in mediating theresponse toward all environmental stresses (Fig. 7, Dand E).The observed increase in salt tolerance exhibited

specifically by Atcaf1a is supported at the molecularlevel by our microarray profiling experiments. Weobserved that 29% of transcripts that increase in abun-dance in nonwounded Atcaf1a-1 plants are genes pre-viously shown to be induced by salt treatment(Ma et al., 2006). This enrichment in salt-inducedgenes is highly significant at a P value of 5.5 3 10211,as calculated by the hypergeometric test (Sokal andRohlf, 1995; Covington and Harmer, 2007).Included in this common gene set is ALDH7B4,

which has been shown to confer salt tolerance whenoverexpressed (Kotchoni et al., 2006). Consistent withAtcaf1b mutants behaving like wild-type plants inresponse to salt treatment, there was no overlap be-tween transcripts increased in Atcaf1b-2 and salt-induced transcripts. The transcript profiling experimentalso provides insight into the increased sensitivity ofboth Atcaf1a and Atcaf1b to MV. One gene, galactinolsynthase2 (GolS2), which is decreased in abundance innonwounded Atcaf1 as well as Atcaf1b, confers toler-

ance to MV when overexpressed (Nishizawa et al.,2008).

In conclusion, we show that the CAF1 family hasexpanded in angiosperms and that a subset of theAtCAF1 family responds to mechanical stress. Signif-icantly, we also demonstrate that both AtCAF1a andAtCAF1b directly or indirectly mediate abiotic stresstolerance. However, they are not functionally redun-dant in all cases, at either the molecular or biologicallevel. We therefore hypothesize that the AtCAF1 fam-ily members may have undergone sub- or neofunc-tionalization subsequent to gene duplication. In thefuture, employment of more sensitive techniques suchas RNA immunoprecipitation followed by sequencingwill be useful in identification of the complete suite ofAtCAF1a and AtCAF1b direct mRNA targets. Addi-tionally, biochemical characterization of AtCAF1a andAtCAF1b associated mRNA-binding proteins willprovide further insight into how these deadenylasesact with such a high degree of specificity.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 plants were grown

in a 16-h light/8-h dark photoperiod at 22�C. All experiments were conducted

on 3-week-old soil-grown plants unless otherwise noted. Atcaf1a-1 (Salk_

070336; Liang et al., 2009), Atcaf1a-2 (Salk_050279), Atcaf1b-2 (Salk_121555),

and Atcaf1b-3 (Salk_044043) T-DNA insertional lines (Alonso et al., 2003) were

obtained from the Arabidopsis Biological Resource Center. For wounding

treatments all rosette leaves were mechanically wounded one to two times

with a hemostat, resulting in approximately 20% of the leaf being damaged, as

previously described (Walley et al., 2007). Mechanical wounding was per-

formed 4 to 6 h after dawn.

BLAST Analyses to Identify CAF1 Homologs in Plants

Unless otherwise stated, all computer analyses were performed on a single

Macintosh iMac running Mac OS 9 or OS X. Using the Arabidopsis CAF1a

(AtCAF1a) sequence as a query we performed a BLAST search to the

Viridiplantae node at Phytozome (www.phytozome.net). After filtering out

any repetitive sequences we downloaded all of the retrieved sequences. Yeast

(Saccharomyces cerevisiae), fly, mouse, and human CAF1 sequences were

identified by performing a BLAST search at the National Center for Biotech-

nology Information. Locally installed versions of Se-Al and CLUSTALWwere

used to generate alignments using all the obtained amino acid sequences,

which were subsequently edited to include only conserved regions of the

proteins.

Phylogenetic Analysis

All steps for phylogeny reconstruction were performed according to

Harrison and Langdale (2006). Using the amino acid sequence alignment

generated above we performed a parsimony and heuristic search to generate a

phylogenetic tree in PAUP* (Swofford, 1999). The 10 trees generated by this

analysis were subsequently computed into a strict consensus tree and boot-

strap values were calculated in PAUP*.

RT-PCR

Total RNA from rosette leaves was isolated by TRIzol extraction (Life

Technologies) and further purified using Qiagen RNeasy kit with on-column

DNase treatment (Qiagen Inc.). RNA was reverse transcribed using Super-

script III (Invitrogen). PCR for RT-PCR were conducted in 25 mL reactions

containing 20 ng cDNA, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.05 mM each



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primer, and 1 unit Choice-Taq blue (Denville Scientific) and amplified for 29

cycles. Quantitative RT-PCR was conducted in 50-mL reactions containing 10

ng cDNA, 13 iQ SYBR Green supermix (Bio-Rad Laboratories), and 200 or 250

nM each primer. Amplification and data analysis were carried out as previ-

ously described (Walley et al., 2008). The internal controls At4g34270 and

At4g26410 previously described were used for transcript normalization

(Czechowski et al., 2005). Primers are listed in Supplemental Table S3.

Microarray Analyses

The Agilent Arabidopsis V3 oliogoarray chip containing 60-mer oligos

probes for 28,500 genes from The Institute for Genomic Research’s ATH1 v.5

annotation as well as 10,000 probes based on Massively Parallel Signature

Sequencing data (Meyers et al., 2004) were utilized for transcript profiling

(Agilent Technologies). Total RNA was prepared from three independent

biological replicates for each genotype 3 treatment combination as described

for RT-PCR. Prior to hybridizations, the quality and quantity of the total RNA

sample was confirmed by running on an Agilent 2100 bioanalyzer, and by

using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies

Inc.). Labeled cRNA was produced using the Agilent low RNA input linear

amp kit according to the manufacturer’s protocol. Briefly, total RNAwas used

as a template for cDNA synthesis primed with oligo(dT) followed by pro-

duction of cRNA labeled with cyanine dyes. Each experimental sample was

labeled with cy3. Additionally, aliquots from each experimental sample were

pooled and labeled with cy5, serving as the reference sample for normaliza-

tion. Labeled cy3 and cy5 cRNAwas then mixed and hybridized according to

Agilent protocol. Arrays were washed and dried, then scanned on an Agilent

DNA microarray scanner. The raw TIFF images were analyzed and normal-

ized using the Agilent Feature Extraction software v. 8.1 using the recom-

mended default settings. The normalized data was then imported into

GeneSpring GX7.3 (Agilent Technologies) to analyze for misexpressed genes

according to the manufacturer’s protocol. Genes with a fold-change$ 2.0 and

a P value # 0.5 were selected as misexpressed.

Comparison of Transcript Profiles

The statistical significance of the observed overlap in transcript profiles, as

well as enrichment of GO terms, was analyzed using hypergeometric tests

(Sokal and Rohlf, 1995; Covington and Harmer, 2007; Rivals et al., 2007).

PAT Assay

PATassays were performed as previously described (Salles and Strickland,

1999) on RNA isolated for qRT-PCR. PCR was conducted in 25 mL reactions

containing 1 mL of PATcDNA as template, 1.5 mM MgCl2, 0.2 mM each dNTP,

0.5 mM each primer, and 1 unit Choice-Taq blue (Denville Scientific) and

amplified for 35 cycles. PCR products were run on a 2% agarose gel and

stained with ethidium bromide for visualization.

Stress-Tolerance Assays

For salt (NaCl) and reactive oxygen species (MV) stress assays seeds were

planted on plates containing 13 Murashige and Skoog basal media with

Gamborg’s vitamins (Sigma Aldrich). Additionally, 200 mM NaCl or 5 mM MV

(Sigma Aldrich) was added to the media as indicated. For salt assays 25 seeds

were planted per plate and each plate was scored for percent germination (n =

4). For MV assays 15 seeds per genotype were plated on each plate (all three

genotypes on the same plate) and each plate was scored for percent survival

(n = 9). Waterlogging experiments were based upon a previous study (Huynh

et al., 2005). Plants for waterlogging were grown in individual pots. Three-

week-old plants were then treated by submerging each pot individually in

water 1 cm above the soil surface. After 7 d the pots were removed from

submersion and plants were given 5 d to recover prior to weighing. Botrytis

cinerea detached leaf assays were performed using the B. cinerea isolates B05.10

and DN (Rowe and Kliebenstein, 2007). Arabidopsis leaves were inoculated

with 5 mL of spores at a concentration of 50,000 spores/mL (Denby et al., 2004;

Rowe and Kliebenstein, 2007; Walley et al., 2008). Statistical differences

between wild type and mutants were detected using t tests.

The microarray data discussed in this publication have been deposited in

the National Center for Biotechnology Information GEO (http://www.ncbi.

nlm.nih.gov/geo/) and are accessible through GEO series accession

GSE17022. AtCAF1a: At3g44260, AtCAF1b: At5g22250, AtCAF1c: At1g27820,

AtCAF1d: At1g27890, AtCAF1e: At1g61470, AtCAF1f: At3g44240, AtCAF1g:

At1g06450, AtCAF1h: At1g15920, AtCAF1i: At5g10960, AtCAF1j: At1g80780,

AtCAF1k: At2g32070, AtPI4Kg3: At5g24240, GolS2: At1g56600, Atcaf1a-1:

Salk_070336, Atcaf1a-2: Salk_050279, Atcaf1b-2: Salk_121555, and Atcaf1b-3:

Salk_044043.

Supplemental Data

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

Supplemental Figure S1. Characterization of Atcaf1a and Atcaf1b Salk

T-DNA alleles.

Supplemental Figure S2. GO annotation of Atcaf1a and Atcaf1b misex-

pressed genes.

Supplemental Figure S3. Normalized microarray expression values of

AtPI4Kg3.

Supplemental Figure S4. Survival of wild type, Atcaf1a-2, and Atcaf1b-3 on

5 mM MV (paraquat).

Supplemental Table S1. List of misexpressed genes in Atcaf1a-1 and

Atcaf1b-2 compared to wild-type plant, at different time points post

wounding.

Supplemental Table S2. Numbers of misexpressed genes in each mutant

relative to wild type at each time point post wounding.

Supplemental Table S3. List of primers used in RT-PCR analyses.

ACKNOWLEDGMENTS

We thank Michael Covington for assistance with the hypergeometric tests.

We would also like to thank Mandy Lee and Jessica Weant for assistance with

the waterlogging experiments.

Received October 7, 2009; accepted November 25, 2009; published December

2, 2009.

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