of the nine cytidine deaminase-like genes in arabidopsis ... · of the nine cytidine deaminase-like...

Of the Nine Cytidine Deaminase-Like Genes inArabidopsis, Eight Are Pseudogenes and Only One IsRequired to Maintain Pyrimidine Homeostasis in Vivo1

Mingjia Chen, Marco Herde, and Claus-Peter Witte*

Leibniz University Hannover, Institute of Plant Nutrition, Department of Molecular Nutrition andBiochemistry of Plants, 30419 Hannover, Germany

ORCID IDs: 0000-0002-0943-3991 (M.C.); 0000-0003-2804-0613 (M.H.); 0000-0002-3617-7807 (C.-P.W.).

CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of Cnucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shownpreviously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed forother members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionalitytests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants onlyrequire a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and justone has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivowas demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine inseeds. Cytidine hydrolysis in cdamutants is likely caused by NUCLEOSIDEHYDROLASE1 (NSH1) because cytosine accumulationis strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicatethat a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction inrosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidinecatabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations.

The catabolism of nucleotides is part of the plantmetabolic network for nutrient remobilization (Zrenneret al., 2009; Werner et al., 2010), which is particularlyimportant for nitrogen (Xu et al., 2012). Pyrimidinenucleotides contain two nitrogen atoms in the ring, andin the case of cytosine, a third amino group nitrogen islinked to the C4 carbon of the heterocycle. The hydro-lysis of this amino group occurs at the nucleoside levelby the deamination of cytidine to uridine mediated byCYTIDINE DEAMINASE (CDA; Fig. 1). By contrast,adenosine is deaminated as nucleoside monophosphate(AMP to IMP by AMP deaminase; Xu et al., 2005),whereas G, similar to C, is deaminated at the level of thenucleoside (guanosine to xanthosine by guanosine de-aminase; Dahncke and Witte, 2013). Cytidine deamina-tion is required to feed C bases into pyrimidine ringcatabolism, which is initiated from uracil (Fig. 1).

There are two classes of CDAs: (1) a tetrameric typeof about 15 kD per subunit represented by the enzymefrom Bacillus subtilis (Johansson et al., 2002) and alsofound in mammals; and (2) a dimeric type of about32 kD per subunit represented by the enzyme fromEscherichia coli (Betts et al., 1994), which is also presentin plants. The dimeric CDAs probably arose from thetetrameric class by gene duplication and subsequentfusion (Faivre-Nitschke et al., 1999). Although thestructural fold of each CDA domain in the dimericCDAs has been conserved, the sequences of these do-mains have diverged. Only the N-terminal domain hascatalytic activity and is able to bind a zinc cofactor(Johansson et al., 2002).

Cytosine in RNA can be subject to deamination aswell, a process called C-to-U editing. In plants, thisprocess occursmainly in chloroplasts andmitochondriainvolving pentatricopeptide proteins for target recog-nition. Which factor catalyzes the deamination is stillnot clear, but certain subtypes of pentatricopeptideproteins containing a C-terminal DYW domain bindzinc and may contribute directly to the deaminatingactivity (Hayes et al., 2013; Shikanai, 2015). Recently,C-to-U editing also was discovered in nuclear planttRNA-Ser(AGA) and tRNA-Ser(GCT), but the biologi-cal function of this modification and the responsibledeaminase are still unknown (Zhou et al., 2014).

ACDAofArabidopsis (Arabidopsis thaliana; At2g19570)has been expressed in E. coli and purified for biochemicalcharacterization (Faivre-Nitschke et al., 1999; Vincenzetti

1 This work was supported by the China Scholarship Council(scholarship grant no. [2012]3013 to M.C.) and the DeutscheForschungsgemeinschaft (grant no. WI 3411/4–1).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Claus-Peter Witte ([email protected]).

M.C. performed all experiments; C.-P.W. and M.H. performed thebioinformatics analyses; C.-P.W. conceived the project; C.-P.W. andM.C. wrote the article.

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et al., 1999). The dimeric enzyme deaminates cytidine (Kmof 150–250 mM, Vmax of 58–60 units mg21) and deoxycyt-idine (Km of 75–120 mM, Vmax of 38–49 units mg21), bindsone zinc ion per subunit, and was suggested to locate tothe cytosol because of the lack of an apparent subcellulartargeting sequence. Faivre-Nitschke et al. (1999) postu-lated the presence of several CDA copies in Arabidopsisbased on Southern-blot data. Sequencing of the Arabi-dopsis genome revealed the presence of a CDA genefamily comprising nine members. These form a phylo-genetic group distinct from other nucleoside or RNA-editing deaminases of this plant (Zhou et al., 2014).Eight members of the Arabidopsis CDA gene family aretightly clustered on chromosome 4, beginning at locusAt4g29570 and ending at locus At4g29650 with one un-related gene (At4g29590) interspersed.

Here, the CDA gene family of Arabidopsis and soy-bean (Glycine max) was analyzed, predicting and test-ing the functionality of different family members. Theanalysis was extended to predict the functionality ofCDA orthologs from other sequenced plants. Usingmutants, we queried whether AtCDA is required andsufficient to maintain pyrimidine homeostasis in vivoand describe the effects of CDA mutation on growth,the pyrimidine metabolite profile, and the resistance totoxic pyrimidine nucleoside analogs.

RESULTS

Bioinformatic Analyses

To determine consensus sequences of highly con-served amino acids in dimeric CDAs, a multiple align-ment of genuine CDA sequences was generated. Usingthe amino acid sequence of one biochemically charac-terized CDA fromArabidopsis (locus At2g19570; Faivre-Nitschke et al., 1999; Vincenzetti et al., 1999) as a query inBLAST searches of the Phytozome database version 9.1accessible on the Internet, CDA sequences from thoseplants were recovered that possess only a single CDA

gene. Assuming that CDA is an indispensable compo-nent of plant primary metabolism, this strategy likelyidentified only truly functional CDAs. Additionally, theConcise Protein Database at the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/genomes/prokhits.cgi), which contains data fromfully sequenced genomes, was searched using the samequery as above but selecting only bacterial sequencescorresponding to CDA of the dimeric type. From thealignment (Supplemental Fig. S1), two consensus se-quences were derived: (1) the overall consensus ofabsolutely conserved amino acids in dimeric CDAs(marked red in the alignment); and (2) the consensus ofabsolutely conserved amino acids in dimeric CDAs ofvascular plants (marked with green triangles above thealignment).

Next, all nine members of the CDA family fromArabidopsis as well as members of the CDA families ofthe closely related speciesArabidopsis lyrata andCapsellarubella were aligned, and the two consensus sequencesderived from Supplemental Figure S1 were annotatedin this alignment (Supplemental Fig. S2; deviationsfrom the overall consensus are marked in yellow).Similarly, CDA sequences of all other plants thatcontain more than one CDA copy in their respectivegenomes were aligned (Supplemental Fig. S3; here,deviations from both consensus sequences are markedin yellow). Deviations from the respective consensusand insertions/deletions were counted in each align-ment and are summarized in Supplemental Tables S1and S2, respectively.

For the CDA gene family of Arabidopsis, these datarevealed that only the CDA encoded at locus At2g19570matches exactly to the general and the plant CDAconsensuses. The other eight proteins (for locus identi-fiers, see Supplemental Table S1) deviate in at least oneand up to 12 positions from the general consensus andin at least nine and up to 24 positions from the plantconsensus. Additionally, these proteins have at leasttwo insertions or deletions not found in other plantCDAs encoded by single-copy genes. All genes withsequence alterationswere expressedweakly or not at allin comparison with the CDA encoding the protein thatmatches the consensus (Supplemental Table S3). Simi-lar data were obtained for the CDA families from A.lyrata and C. rubella. This suggested that each of thesethree species possesses only one functional copy ofCDA, whereas the other genes had accumulated mu-tations that likely resulted in the loss of CDA activity.

The eight CDA family members with deviationsfrom the CDA consensuses were called CDA-LIKE1(AtCDAL1) to AtCDAL8 (or Ath-L1 to Ath-L8 in theSupplemental Data), and the one CDA matching theconsensuses was called AtCDA without numbering.This nomenclature was analogously applied for theCDA families from A. lyrata and C. rubella.

The sequence analysis was extended to the nine otherplant species in Phytozome version 9.1, which con-tained more than one CDA gene (Supplemental Fig. S3;Supplemental Table S2). By the same criteria outlined

Figure 1. Model of cytosolic pyrimidine catabolism and salvage inArabidopsis. kinase, Nucleoside kinase; PPase, phosphatase acting on59 monophosphate nucleotides (59 nucleotidase). Dashed connectionsare only relevant in cda mutants.

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above, probable nonfunctional CDAs were identified.Judged by sequence, in only five species out of 32 vas-cular plants analyzed were more than one fully func-tional CDA identified.If AtCDAL1 to AtCDAL8 lost function, one would

postulate that the number of mutations resulting in adifferent amino acid sequence (nonsilent) observed forthe corresponding genes would be far higher than forAtCDA, which is subject to conserving selection. Usingthe sequence data of 812 Arabidopsis accessions, thequantity of independent mutations was compared forthese genes and expressed as silent and nonsilent mu-tations on the protein level. Incidents of 1-bp deletionsresulting in frameshift mutations in each gene werecounted as well (Table I). Indeed, mutational rates werestrongly increased for AtCDAL1 to AtCDAL8 in com-parison with AtCDA, reinforcing the notion that theseare pseudogenes.

Enzymatic Activity

To validate the results from the bioinformatic anal-yses,AtCDA,AtCDAL3, andAtCDAL4were cloned, thecorresponding proteins were transiently expressed inNicotiana benthamiana as C-terminally tagged StrepIIvariants and affinity purified, and the specific activityfor cytidine and deoxycytidine was assessed. AtCDAL3and AtCDAL4 were selected because they show com-paratively few deviations from the general and plantconsensuses (Supplemental Table S1). We failed toamplify AtCDAL3 and AtCDAL4 from leaf comple-mentary DNA (cDNA) but were able to obtain clonesfrom genomic DNA. Transient expression and pu-rification of the corresponding proteins resulted inhighly pure preparations for each enzyme (Fig. 2;Supplemental Fig. S4) used for the determinationof kinetic constants. The catalytic efficiencies ofAtCDA were 48.3 and 85.9 mM

21 s21 for cytidine anddeoxycytidine, respectively. For the same substrates,AtCDAL4 showed catalytic efficiencies of 0.02 and1.7 mM

21 s21, respectively (Table II; Supplemental

Fig. S5), whereas for AtCDAL3, no activity could bedetected. Surprisingly, AtCDAL4, with one deviationfrom the general consensus and 11 deviations from theplant CDA consensus (Supplemental Table S1), hadnot completely lost activity. Nonetheless, the kineticparameters of AtCDAL4 (Table II) are very likely in-sufficient for a function as cytidine/deoxycytidine de-aminase in vivo.

The soybean genome contains three full-length CDAgenes potentially encoding the proteins GmCDA1 toGmCDA3. GmCDA3deviates in two positions from thegeneral and in seven positions from the plant CDAconsensus and has a longer sequence deletion affectingamino acids absolutely conserved in dimeric CDAs(Supplemental Fig. S3; Supplemental Table S2). There-fore, it is highly unlikely that GmCDA3 represents afunctional CDA. GmCDA1 and GmCDA2 both deviatefrom the plant consensus in several positions. Moststrikingly, GmCDA1 translated from the soybeangenome sequence contains a Y at position 164 thatis conserved as N in all other plant sequences, andGmCDA2 contains F at position 65 where normally L isconserved in plants. These nonconservative exchangesmight compromise the activity of the respective pro-teins, which would reduce the capability of soybean tocatalyze cytidine deamination. To test this hypothesis,the coding sequences of both proteins were cloned fromsoybean leaf cDNA. To our surprise, we found an Nand not a Y codon in the cDNA coding for amino acid164 of the GmCDA1 protein (codon UAC instead ofAAC, as found in the genome sequence). Because N isabsolutely conserved in plant CDAs, we concluded thatthe soybean genome sequence must be wrong at thisposition. Nonetheless, GmCDA1 and GmCDA2 weretransiently expressed and purified (Supplemental Fig. S4)and the activity was assessed. GmCDA1 had catalyticefficiencies of 101.5 and 269.09 mM

21 s21 for cytidine anddeoxycytidine, respectively. The catalytic efficiencies ofGmCDA2 were 39.4 and 178.3 mM

21 s21 for the samesubstrates (Table II). It is possible that the nonconservativeL-to-F exchange of amino acid 65 is mainly responsible for

Table I. Analysis of mutational frequency in AtCDA and AtCDAL genes of different Arabidopsis accessions

For each CDA isoform, the number of independent single-nucleotide polymorphisms (SNPs) detected in812 Arabidopsis ecotypes from the 1001 Genome Project resulting in either the same amino acid as in thereference (syn/100aa) or a different amino acid/stop codon (nonsyn/100aa) is shown normalized to thedifferent protein lengths (SNPs per 100 amino acids). The number of deletions resulting in frameshifts isgiven in addition to the amino acid position(s) affected by the deletion. SNPs supported by the sequence ofonly one ecotype are not included.

Gene Encoded Protein syn/100aa nonsyn/100aa No. of Frameshifts

At2g19570 AtCDA 4.7 2.7 –At4g29570 AtCDAL1 4.4 16.0 1 (Asp-175)At4g29580 AtCDAL2 2.9 10.6 2 (Trp-227 and Glu-420)At4g29600 AtCDAL3 7.2 17.6 –At4g29610 AtCDAL4 6.8 5.5 –At4g29620 AtCDAL5 4.2 17.8 2 (Glu-221 and Leu-317)At4g29630 AtCDAL6 6.3 9.4 1 (Leu-68)At4g29640 AtCDAL7 9.2 20.5 –At4g29650 AtCDAL8 4.0 7.2 –

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the comparatively low activity of GmCDA2. The equiv-alent residue lines the active site pocket in the enzymes ofE. coli (Betts et al., 1994) and Vibrio cholera (Protein DataBank accession no. 4EG2) and is conserved as L, I, V, orMin CDAs of plants and bacteria (Supplemental Fig. S1).For soybean, GmCDA1, lacking this nonconservativeexchange, is the most active CDA isoform. The relatedspecies Phaseolus vulgaris and Medicago truncatula alsopossess a CDA isoform with this L-to-F exchange(PvCDA2 and MtCDA2; Supplemental Fig. S3), indicat-ing that, in these plants, the other respective CDA iso-forms, PvCDA1 and MtCDA1, which agree with theconsensus at this position, are the most active enzymes.

Subcellular Localization

To clarify the subcellular location of AtCDA andthe two soybean CDAs, fusion proteins comprising aC-terminal yellow fluorescent protein (YFP) tag (AtCDA-YFP, GmCDA1-YFP, and GmCDA2-YFP) were tran-siently expressed in leaves ofN. benthamiana. As a control,cytosolic b-ureidopropionase fused to cyan fluorescent

protein (CFP) from Arabidopsis (b-UP-CFP; PYD3-CFP)was coexpressed (Zrenner et al., 2009). Additionally,transgenic Arabidopsis plants expressing AtCDA-YFPwere generated and leaf protoplasts were prepared.Confocal microscopy revealed that AtCDA is located inthe cytosol after transient expression (Fig. 3, A–C) as wellas in leaf protoplasts from the transgenic line (Fig. 3, D andE). The stability of the transgene was demonstrated byan immunoblot (Fig. 3F). A cytosolic location also wasfound for the two soybean CDAs (Supplemental Figs.S6 and S7).

Deamination of Cytidine in Vivo

The bioinformatic analyses (Table I; SupplementalFig. S2; Supplemental Table S1) and the biochemicalassessment of two members of the Arabidopsis CDAfamily (Table II) suggested that AtCDA is the onlycytidine-deaminating enzyme in this plant. Two inde-pendent plant lines with homozygous transfer DNA(T-DNA) insertions in theAtCDA genewere isolated fromthe mutant collection of the German Plant GenomicsResearch Program (cda-1 [GK645H07]; Kleinboeltinget al., 2012) and the Salk Institute Genomic AnalysisLaboratory (cda-2 [SALK_036597]; Alonso et al., 2003).

The insertion positions were mapped to 39 of base 96for cda-1 and to 39 of base 150 for cda-2 when countedfrom the start codon (Fig. 4A). For both mutant lines,the absence of intact mRNA was confirmed by reversetranscription-PCR (Fig. 4B).

In wild-type seeds of Arabidopsis, only a smallamount of cytidine was detected by HPLC. By contrast,in seeds of both mutant lines, an accumulation of cyti-dine (60 times higher than the concentration in the wildtype), cytosine, CMP, and also some uridine was ob-served (Fig. 4, C and D). The metabolites were identifiedby retention time and UV spectra of the correspondingstandards. The introduction of anAtCDA-YFP transgenedriven by the 35S promoter into the cda-1 backgroundsuppressed the accumulation of these metabolites (Fig.4C, bottom).

In leaves, cytidine and cytosine accumulated in themutant lines in an age-dependent manner but to lowertotal concentrations than in seeds (CMP and uridinewere not detected). Such an accumulation was not ob-served in the wild type or the complementation line(Fig. 5). The higher metabolite accumulation in seeds incomparison with leaves corresponded well to a signif-icantly higher transcript amount of CDA in repro-ductive organs, especially siliques, compared withleaves and other tissues (Supplemental Fig. S8).

Figure 2. Purification of C-terminally StrepII-tagged AtCDA from leafextracts of N. benthamiana with Streptactin affinity chromatography.Samples (10 mL) were taken at different stages of the purification andvisualized by SDS gel electrophoresis followed by colloidal CoomassieBlue staining (A), silver nitrate staining (only elution fraction; B), andwestern blotting with Streptactin-alkaline phosphatase (AP) conjugatedetection (C). Lane 1, Extract of soluble proteins; lane 2, proteins notbound after incubationwith Streptactin affinitymatrix; lane 3, protein inthe last wash fraction; lane 4, pool of eluted protein; lane 5, protein lefton the matrix after elution, released by boiling in SDS buffer.

Table II. Kinetic parameters for substrates of recombinant CDAs

kcat, Turnover number.

SubstrateAtCDA AtCDAL4 GmCDA1 GmCDA2

Km kcat kcat/Km Km kcat kcat/Km Km kcat kcat/Km Km kcat kcat/Km

mM s21 mM21 s21 mM s21 mM

21 s21 mM s21 mM21 s21 mM s21 mM

21 s21

Cytidine 0.35 6 0.07 17.01 6 0.87 48.3 28.93 6 1.76 0.50 6 0.01 0.02 1.37 6 0.17 139.01 6 4.44 101.5 3.02 6 0.29 118.97 6 2.97 39.4

Deoxycytidine 0.11 6 0.02 9.70 6 0.34 85.9 3.74 6 0.35 6.38 6 0.17 1.7 0.24 6 0.01 64.85 6 0.94 269.1 0.33 6 0.02 58.84 6 0.94 178.3


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These data demonstrate that AtCDA is the centralcytidine-deaminating enzyme in vivo, despite thepresence of another eight CDAL genes in Arabidopsis.The accumulation of deoxycytidine was not observed.Either DNA degradation was very limited or an alter-native deaminase, such as AtCDAL4, might havecompensated for the loss of AtCDA with respect todeoxycytidine deamination in the mutant lines. Wefavor the first explanation because (1) AtCDAL4 has avery low catalytic efficiency (Table II) and (2) an ac-cumulation of deoxyguanosine was not observed inguanosine deaminase knockout lines despite strongaccumulation of guanosine (Dahncke and Witte, 2013),indicating that no significant DNA degradation occurs.

The Origin of Cytosine in the cda Mutant

The cda-1 mutant was crossed to the mutant ofNUCLEOSIDE HYDROLASE1 (NSH1; mutant allelensh1-1 [SALK_083120]; Jung et al., 2011).NSH1 catalyzesthe hydrolysis of uridine (Fig. 1) or xanthosine to riboseand the respective nucleobases uracil or xanthine. We hy-pothesized that NSH1 also might degrade cytidine to cy-tosine and ribose in the cda background, explaining theaccumulation of cytosine in this mutant. It has been re-ported that cytidine is not a substrate for NSH1 (Junget al., 2009).However, at the high cytidine concentration inthe cdamutant and during the long exposure times of theenzyme to the potential substrate in vivo, even catalysis at

a very low rate might be enough to partially destabilizecytidine. In the cda-1 nsh1-1 double mutant, cytosine ac-cumulation was reduced by 78% compared with theconcentration observed in the cda-1 single mutant (Fig.6B), consistent with the idea that NSH1 can hydrolyzecytidine in vivo if this metabolite accumulates. An alter-native explanation is that the high uridine concentration inthe nsh1 background prevents an unknown nucleosidehydrolase from using cytidine as a substrate. Interest-ingly, the decrease of cytosine did not lead to a corre-sponding increase in cytidine concentration in the doublemutant, possibly because the cytidine pool also can betapped by a kinase generating CMP (Fig. 1).

The slight increase of uridine in the cda mutantcompared with the wild type (Fig. 4, C and D), which atfirst seems counterintuitive because uridine is the pro-duct of the CDA reaction (Fig. 1), might be explained byan occupation of NSH1 with the abundant cytidine,thereby partially preventing NSH1-catalyzed uridinehydrolysis.

The nsh1mutant accumulated uridine and also someUMP in the seed (Fig. 6, A and B; Dahncke and Witte,2013), as was shown previously for nonseed tissues(Jung et al., 2009; Riegler et al., 2011). The uridine con-centration is about half in the double mutant in com-parison with the nsh1 mutant (Fig. 6B), indicating thatcytidine deamination contributes 50% to the uridinepool in seeds of the nsh1 mutant. The UMP accumula-tion observed in the nsh1 mutant background is de-creased and the CMP accumulation observed in the cdamutant background is slightly increased in the doublemutant (Fig. 6).

The Susceptibility to Toxic Nucleoside Analogs Is Alteredin cda Plants

Nucleoside analogs are frequently used in cancertherapy, because they are cytotoxic after incorporationinto the cellular nucleotide pool that is used for DNAreplication (Jordheim et al., 2013). Cytidine deaminaseis a well-recognized factor in altering the effects of nu-cleoside analogs on human cells (Serdjebi et al., 2015).

To investigate the impact of CDA in plants on thetoxicity of the nucleoside analogs 5-fluorocytidine(5-FC) and 5-fluorouridine (5-FD), cda-1, cda-2, Col-0,and the complementation line (cda-1 + AtCDA-YFP)were grown on one-half-strengthMurashige and Skoogmedium supplemented with these compounds. Thegrowth of cda-1 and cda-2 plants was blocked shortlyafter germination by 5 mM 5-FC, whereas Col-0 and thecomplementation line grew only slightly slower thanthe control (Fig. 7). The situation was reverse on me-dium supplemented with 50 mM 5-FD. The mutant linesgrew better than Col-0 and the complementation line(Fig. 7). Arabidopsis shows a lower tolerance to 5-FCthan to 5-FD. The mutation of CDA decreases the 5-FCtolerance even further but increases the 5-FD tolerance.It appears that 5-FC is easily salvaged by a nucleosidekinase and that the extent of salvage is increased in thecda mutant, because the catabolic route is blocked in this

Figure 3. Subcellular localization of AtCDA. A to C, Confocal fluo-rescence microscopy images of cells at the lower leaf epidermis of N.benthamiana transiently coexpressing C-terminally AtCDA-YFP andb-UP-CFP fusion proteins. YFP (A), CFP (B), and YFP and CFP (C) de-tection are shown. Bars = 30 mm. D and E, Mesophyll cell protoplasts ofcda-1 plants transformed with AtCDA-YFP. YFP (D) and YFP andchlorophyll (E) detection are shown. Bars = 10mm. F, Stability test of theAtCDA-YFP fusion protein from transgenic Arabidopsis plants analyzedby an immunoblot developed with a GFP-specific antibody.





genetic background. The increased CMP pool in the cdamutant (Fig. 6B) also suggests that cytidine salvage isboosted. 5-FD is salvaged as well, and the strongertolerance of the cda mutant to this compound might beexplained by a high occupancy of the nucleoside kinasewith the excess cytidine in this mutant, which leavesless kinase capacity for 5-FD salvage. Interestingly,these data indicate that pyrimidine nucleosides areprobably salvaged by kinases with dual specificity foruridine and cytidine.

The Mutation of CDA Reduces Plant Performance

Both cda mutant lines displayed decreased growthand produced fewer leaves compared with the wildtype or the complementation line (Fig. 8). The effectbecomes apparent at about 26 d after germination un-der long-day conditions (16 h of light) when quantifiedby measuring the rosette diameter. Partially compro-mised pyrimidine catabolism or the interference of theaccumulating intermediates with metabolism might bethe reasons for the reduced plant performance. Becausesuch a drastic growth depression is not observed inother pyrimidine catabolism mutants, such as the nsh1mutant (Jung et al., 2011; Riegler et al., 2011) or the pydmutants (Zrenner et al., 2009), the interference of ac-cumulating pyrimidine compounds with metabolismappears to be the most plausible explanation for thegrowth depression observed in the cda mutants. How-ever, in reproductive organs and during embryo devel-opment, where the highest accumulation of pyrimidinecatabolism intermediates (Fig. 5) and the highest ex-pression of CDA (Supplemental Fig. S8) were detected,no phenotypic differences between the wild type, themutants, and the complementation line were observed(Supplemental Fig. S9).

DISCUSSION

Although Arabidopsis possesses a family of ninegenes potentially encoding cytidine deaminases, onlyone of these genes gives rise to a fully functional en-zyme: AtCDA. What is the function of the other eightCDAL genes? It has been speculated that members ofthe CDA family might be involved in a putative cellularmachinery that introduces hypermutations into viralgenomes as a means of antiviral defense (Lin et al.,2009). Alternatively, CDAL proteins might have afunction in C-to-U editing in the mitochondria andplastids or in the recently discovered C-to-U editing of

Figure 4. Genetic characterization and seed pyrimidine metaboliteprofiles of two independent cdamutant lines. A, Genomic organizationof the At2g19570 locus and positions of the T-DNA insertions (triangles)in cda-1 (GK645H07) and cda-2 (SALK_036597C). The box representsthe coding sequence, which does not contain any introns. Approximateprimer positions for N261, 448, N61, and N262 are indicated. B, PCRand reverse transcription-PCR analyses of homozygous cda-1 and cda-2

lines and the wild type Columbia-0 (Col-0). C, Analysis of seed me-tabolite extracts using HPLC with photometric detection of the wildtype, the two cda mutants, and a cda-1 line complemented with anAtCDA-YFP transgene. mAU, Milliabsorption units. D, Quantificationof metabolites accumulating in mutant seeds. Error bars indicate SD (n =4). Different letters mark significant differences at P , 0.05. fw, Freshweight; n.d., not detectable.


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two nuclear plant tRNAs (Zhou et al., 2014). However,there are several arguments against a functional role ofCDAL proteins in these processes. (1) The expansion ofthe CDA family is limited to Arabidopsis and a fewother closely related Brassicaceae members. Dependingon the Brassica spp., the number of CDA family mem-bers varies, arguing that there are at least some nones-sential genes (Supplemental Fig. S2). In line with this,there are some members that are not expressed in anytissue of Arabidopsis according to publicly availableexpression data (Supplemental Fig. S3), and in someArabidopsis accessions, certain CDAL family membershave suffered frameshift mutations (Table I). (2) Mostplants have only one CDA gene and lack CDAL genes.Therefore, it is unlikely that C-to-U editing, which isfound everywhere in the plant kingdom, is performedby CDAL proteins only in the Brassicaceae. (3) Mostediting occurs in mitochondria and plastids, butAtCDA is cytosolic, and there are no predicted target-ing peptides in the CDAL proteins. It also has beenshown that AtCDA does not bind RNA (Faivre-Nitschke et al., 1999). (4) Only AtCDAL4 retains weak

deoxycytidine-deaminating activity. AtCDAL3 is notactive, and the others are so highly mutated that theywill not have maintained activity, especially because inevery protein except AtCDAL3 and AtCDAL4, ab-solutely conserved residues are affected, which wereshown to be directly involved in zinc or substratebinding in the CDA from E. coli (Supplemental Fig. S2;Betts et al., 1994). The inability of the CDAL proteins tocompensate for the loss of CDA in the cda mutant is inline with these observations. (5) The high number ofSNPs in CDAL genes of different Arabidopsis ecotypesresulting in amino acid changes of the correspondingproteins indicates that they are subject to low selectivepressure, which is typical for nonfunctional genes. TheCDAL genes are located in a cluster on chromosome 4.The equivalent region in Brassica rapa still contains aCDA gene, which is very likely functional (BrCDA2;

Figure 5. Cytidine andcytosinecontents in leavesduringplant development.Quantification of cytidine (top) and cytosine (bottom) is shown in rosetteleaves of Col-0, cda-1, cda-2, and the complementation line from 15 to 55 dafter germination (dag). Error bars indicate SD (n = 4). Different letters marksignificant differences at P, 0.05. fw, Fresh weight; n.d., not detectable.

Figure 6. Pyrimidine metabolite analysis of seeds of the cda-1 nsh1-1double mutant in comparison with the respective single mutants andthe wild type. A, HPLC spectrophotometric traces of nsh1-1 and cda1nsh1-1 seed extracts. mAU, Milliabsorption units. B, Quantification ofpyrimidine metabolites in seed extracts of Col-0, cda-1, nsh1-1, and thedouble mutant. Error bars indicate SD (n = 4). Different letters marksignificant differences at P , 0.05. n.d., Not detectable.








Supplemental Table S2), in addition to the BrCDA1 gene.Such a situation also is found in Aethionema arabicum,belonging to the most basal group of the Brassicaceae(Kagale et al., 2014). This indicates that a duplication ofCDA early in Brassicaceae evolution created twogenomicCDA loci. In some crucifers, both loci were functionallymaintained, maybe stabilized by differential expressionpatterns, and in others, one locus lost functionality, cre-ating CDAL genes. In summary, it can be concluded thatAtCDAL1 to AtCDAL8 are most likely pseudogenes, atleast with respect to a C-deaminating function.

In soybean, many genes are present in multiplecopies that, in other plants, are only required in a singlecopy (Polacco et al., 2011; Werner et al., 2013). Soybeanis a paleopolyploid plant whose genome underwentpolyploidization at least twice, the last time relativelyrecently (Kim et al., 2009). This explains the increasednumber of gene copies that also affected CDA. How-ever, GmCDA1 is the most active isoform, whereasGmCDA2 already has suffered critical mutations re-ducing its catalytic efficiency (Table II). It is possiblethat GmCDA2 fulfills a tissue-specific role, or will belost over time, or will acquire a new function.

Cytidine accumulation in seeds of the cdamutant hassecondary effects increasing CMP, cytosine, and uri-dine pool sizes. Cytosine is likely generated by cytidine

hydrolysis via NSH1, which in the wild type would notoccur because cytidine is of low abundance and not agood substrate for NSH1 (Jung et al., 2009). The CMPpool size is probably influenced by the respective ratesof (1) cytidine phosphorylation, likely increased by highsubstrate availability, and (2) CMP dephosphorylation,which might be inhibited by high cytidine concentra-tions. Even more CMP accumulates in seeds of the cdansh1 double mutant, which in addition to cytidine alsoaccumulates uridine (Fig. 6B). A possible explanation isa regulatory role of uridine on the kinase/phosphatasemodule (Fig. 1), leading to the promotion of mononu-cleotide formation. A central regulatory role of theuridine-degrading enzyme NSH1 has been discussed(Jung et al., 2009), which might be mediated by uridineabundance. By contrast, UMP concentrations are sig-nificantly lower in the double mutant than in the nsh1single mutant. This finding might be explained by de-creased substrate availability to the kinase and/orreduced inhibition of the phosphatase, because theuridine pool size also is reduced by 50% in the doublemutant compared with nsh1. Additionally, if uridinehas a regulatory function stimulating mononucleo-tide formation, as hypothesized above, a reduction in

Figure 7. Growth responses to 5-FD and 5-FC. A, Scheme of seedplacement on agar plates with four partitions. B, Col-0, cda-1, cda-2,and the complementation line after 10 d of growth on standard mediumunder long-day conditions (16 h of light). C, Like B but in the presence of5 mM 5-FC, recorded on day 15. D, Like B but in the presence of 50 mM

5-FD, recorded on day 27.

Figure 8. Growth phenotypes of cda lines. A, Rosette diameters of Col-0,cda-1, cda-2, and the complementation line (n = 10 for each) grown45 d under long-day conditions (16 h of light). B, Rosettes and rosetteleaves of the different genotypes at 45 d after germination (dag). Fordocumentation, the inflorescences were removed. Bars = 5 cm.


Chen et al.




uridine concentration would lead to decreased mono-nucleotide levels. In this model, uridine rather has apositive effect on the activity of the nucleoside kinase(s)than a negative effect on the nucleotide phosphatase(s),because there is no CMP accumulation in the nsh1mutant. A further explanation for the reduced UMPconcentrations in the double mutant could be that thenucleoside kinase is more occupied with cytidinephosphorylation and not fully available to uridine inthis mutant. That cytidine can successfully compete forthe nucleoside kinase is indicated by the weak pheno-type of the cda mutants in comparison with the wildtype in the presence of fluorouridine (Fig. 7). Together,these data suggest that the cytosolic nucleoside kinase(s)have dual specificity for uridine and cytidine andmay be stimulated by uridine. Two plastidic uridinekinases (UKL1 and UKL2) from Arabidopsis have al-ready been characterized (Chen and Thelen, 2011), butwhether cytidine is a substrate has not been assessed.An additional three genes with high similarity to uri-dine kinases (UKL3, UKL4, and UKL5) are found in theArabidopsis genome (Mainguet et al., 2009), but theyhave not been functionally characterized yet. However,a biochemically characterized uridine kinase of maize(Zea mays) was shown to have dual specificity for uri-dine and cytidine (Deng and Ives, 1975).It has been claimed that CDA is involved in the

age-related resistance response of Arabidopsis (Carvielet al., 2009), because plants compromised in CDAshowed reduced resistance to the virulent bacteriumPseudomonas syringae pv tomato and to the downy mil-dew fungus Hyaloperonospora parasitica. Additionally,CDA was transcriptionally induced in leaves by Pseu-domonas spp. infection. However, it is shown here thatcda mutants are compromised in their growth, likelydue to the accumulation of metabolites, either cytidinedirectly or its derivatives, at toxic concentrations. Thismetabolic stress might well explain why the cda mu-tants are more susceptible to pathogens, and a specificrole of CDA in age-related resistance does not need tobe postulated. The transcriptional up-regulation ofCDA in this context might be caused by senescence in-duction due to pathogen infection. Public expressiondata as well as our transcriptional data (SupplementalFig. S8) show that CDA transcript amounts increase insenescence.

MATERIALS AND METHODS

Plant Material and Cultivation

Arabidopsis (Arabidopsis thaliana) ecotype Col-0 was chosen as the wildtype. T-DNA insertion mutants of Arabidopsis from the Gabi-Kat collection(GK645H07, cda-1; Kleinboelting et al., 2012) and the SALK collection(SALK_036597, cda-2; and SALK_083120, nsh1-1; Alonso et al., 2003) were or-dered from the European Arabidopsis Stock Centre. Arabidopsis and Nicotianabenthamiana plants were cultivated under the same condition described before(Witte et al., 2004, 2005). Transient expression inN. benthamianawas performedas described by Werner et al. (2008).

To determine alterations in rosette diameter and leaf number, plants of thedifferent genotypes were grown randomly distributed on growth trays andanalyzed for morphological characteristics and rosette diameter. For each

genotype, 10 replicates were grown. Embryo preparation and microscopy wereperformed as described by Hauck et al. (2014).

Agar plates were prepared with one-half-strength Murashige and Skoognutrients supplemented with the toxic nucleoside analog 5-FC or 5-FD whereindicated. Plates were incubated in a controlled growth chamber under long-day conditions (16 h of light at 150 mmolm22 s21, 20°C day and 18°C night, 16%relative humidity).

Cloning, Reverse Transcription, and Real-Time PCR

RNA from plants was prepared using TRI reagent (Sigma) and treated withDNase I (Sigma) following the manufacturer’s instructions. Reverse transcrip-tion using 1 mg of total RNA was performed with Moloney murine leukemiavirus reverse transcriptase (Invitrogen) and a poly(T) primer. PCR employedthe following primers: for AtCDA, N354 + N355; for AtCDAL4, N466 + N467;for AtCDAL3, P1 + P2; for GmCDA1, N363 + N364; and for GmCDA2, N365 +N366. Real-Time PCR employed the following primers: for ACTIN2, 2531 +2532; for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 39, 2527 + 2528;for GAPDH 59, 2529 + 2530; and for AtCDA, N515 + N516.

Protein Purification and Enzymatic Assays

StrepII-tagged AtCDA was affinity purified after transient expression in N.benthamiana as described by Werner et al. (2008). Purified protein was quanti-fied using the Bradford reagent from Serva using bovine serum albumin as astandard. For the standard enzymatic assays, recombinant CDA solutions wereadjusted to the following concentrations using elution buffer of the purificationprocedure: AtCDA at 5 mg mL21, GmCDA1 and GmCDA2 at 1 mg mL21, andAtCDAL4 at 20 mg mL21. For individual reactions, 75 mL of the substrate so-lution was incubated at 30°C for 5 min. The reaction was started by adding25 mL of enzyme solution. In a time course, 20-mL aliquots were withdrawn andadded to 80 mL of water, followed by the addition of 25 mL of phenol nitro-prusside reagent and 50 mL of hypochlorite reagent for colorimetric ammoniaquantification (Witte and Medina-Escobar, 2001). The absorbance was deter-mined by a photometric measurement at 636 nm. Ammonium standard curveswere generated by adding elution buffer instead of enzyme solution to the re-action mix and placing 20-mL aliquots in 80-mL NH4Cl solutions of differentconcentrations prior to detection.

The kinetic constants were determined at 0.1, 0.25, 0.5, 1, 3, and 5 mM cytidineor 0.05, 0.1, 0.25, 0.5, 1, and 2 mM deoxycytidine for AtCDA; 5, 10, 25, 50, 100,200, and 500 mM cytidine or 1, 2.5, 5, 10, 20, and 50 mM deoxycytidine forAtCDAL4; 0.5, 1, 2.5, 5, 10, and 20 mM cytidine or 0.1, 0.25, 0.5, 1, 2.5, and 5 mM

deoxycytidine for GmCDA1; and 1, 3, 5, 10, 20, and 40 mM cytidine or 0.1, 0.25,0.5, 1, 2.5, and 5 mM deoxycytidine for GmCDA2. Kinetic curves were recordedin three to four independent repeats, and kinetic constants were determinedby fitting the data to the Michaelis-Menten equation using Graph Pad Prismsoftware.

Mutant Characterization

Homozygous mutant lines were screened out of a segregating population byPCR using primers 448 + N262 and N261 + N262 for cda-1, primers N61 + N261and N261 + N262 for cda-2, and primers 488 + 2616 and 1905 + 2616 for nsh1-1(Supplemental Table S4). Double mutants were obtained by crossing nsh1-1(male) and cda-1 (female). The PCR products from the mutants were cloned andsequenced to map the exact positions of the insertions.

Todetermine the amount of gene-specificmRNA in themutants, cDNA fromseedlings was prepared as described above. The PCR employed primers N261 +N262, giving rise to a product of 578 bp from the wild-type allele. In bothmutant lines, the T-DNA insertions are flanked by these primers. For the am-plification of ACTIN2 as a control, primers 2531 + 2532 were used. The ex-pression level of NSH1 in the nsh1-1 mutant was determined before by Junget al. (2011).

Metabolite Analyses

Seedlings were ground in liquid nitrogen using amortar, 150mgwas passedfrozen into a 1.5-mL micocentrifuge tube, and 360 mL of cold 0.5 M HClO4 wasadded followed by grinding with a rotating pestle. Samples were incubated onice for 10 min and centrifuged (15 min, 20,000g, and 4°C), and the supernatantwas mixed with 20 mL of alkaline potassium carbonate solution (5 M KOH and2 M K2CO3) to precipitate the perchlorate. After incubation for 5 min on ice,








samples were centrifuged (15 min, 20,000g, and 4°C), and supernatants werefrozen in liquid nitrogen, thawed, centrifuged again as before, and the newsupernatants were transferred to HPLC sample vials. Seed extraction wasperformed by grinding 10 mg of material in 150 mL of HClO4 with a rotatingpestle. The supernatant was removed after centrifuging (15min, 20,000g, and 4°C),and the extractionwas repeated. Both supernatants were pooled and incubated onice for 10min and thenmixedwith 15mL of alkaline potassium carbonate solution.All subsequent stepswere performed as described above. Cytosine, cytidine, CMP,uridine, and UMP (50, 100, 250, 500, 1,000, and 2,000 mM solutions) were treated asthe samples and used as standards. The HPLC analysis procedure followed wasaccording to the description by Dahncke and Witte (2013).

Protoplasting and Subcellular Localization

For protoplasting, two 12-mm leaf discs were cut fromN. benthamiana plantsafter 3 d of transient protein expression. The discs were washed using sterilewater and wounded slightly at the lower surface with a sharp blade. After in-cubation with 500 mM mannitol for 1 h, the discs were transferred into 1 mL ofenzyme solution (500 mM mannitol, 10 mM CaCl2, 5 mM MES/KOH, pH 5.5, 3%Cellulase Onozuka, and 0.75% Macerozyme [Yakult Pharmaceutical]) andvacuum infiltrated at less than 25 mbar for 5 min. Protoplasts were isolated byslow shaking in darkness for 3 h.

For subcellular localization, AtCDA-YFP, GmCDA1-YFP, GmCDA2-YFP,and b-UP-CFP were coexpressed and analyzed by confocal microscopy as de-scribed by Dahncke and Witte (2013).

Mutational Rate and Gene Expression Analyses

Data for SNPs from 812 ecotypes from all available collectionswere obtainedfrom http://1001genomes.org. Whenever possible, the data set with the morestringent SNP-calling parameters was chosen, and data files were reformattedusing in-house scripting for annotation by SNPeff (Cingolani et al., 2012) usingThe Arabidopsis Information Resource 10 Arabidopsis gene model as a refer-ence. Results were filtered for CDA isoforms, separated into synonymous andnonsynonymous SNPs, and counted using in-house scripting.

Short read data sets were downloaded from the National Center for Bio-technology Information short read archive available at http://www.ncbi.nlm.nih.gov/sra and mapped against The Arabidopsis Information Resource 10Arabidopsis gene model using the CLC genome bench (Qiagen). Only readsmapping uniquely to one location were considered and normalized against thetotal number of mapped reads and the length of the respective CDA transcript.

Statistical Analyses

ANOVA followed by Tukey’s honestly significant difference test were per-formed for statistical evaluation. Significance levels of P, 0.05, P, 0.01, and P,0.001 are indicated in thefiguresby single, double, and triple asterisks, respectively.Different letters represent significant differences at the P , 0.05 significance level.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Multiple alignment of CDA sequences fromplants, algae, and bacteria.

Supplemental Figure S2. Multiple alignment of sequences of the CDAfamilies from Arabidopsis and its close relatives A. lyrata and C. rubellafrom the Camelineae tribe.

Supplemental Figure S3. Multiple alignment of CDA from Arabidopsiswith CDA protein sequences from several plants possessing more thanone CDA gene copy.

Supplemental Figure S4.Affinity-purified AtCDAL3, AtCDAL4, GmCDA1,and GmCDA2.

Supplemental Figure S5. Determination of kinetic constants for AtCDA,AtCDAL4, GmCDA1, and GmCDA2.

Supplemental Figure S6. Subcellular localization of GmCDA1.

Supplemental Figure S7. Subcellular localization of GmCDA2.

Supplemental Figure S8. Tissue-specific expression level of AtCDA.

Supplemental Figure S9. Comparison of the phenotypes of reproductiveorgans and embryos of the wild type, cda-1, cda-2, and the complemen-tation line.

Supplemental Table S1. Quantitative evaluation of the deviation from theconsensus sequences derived from the protein alignment in SupplementalFigure S2.

Supplemental Table S2. Quantitative evaluation of the deviation from theconsensus sequences derived from the protein alignment in SupplementalFigure S3.

Supplemental Table S3. Expression analysis of CDA isoforms based onRNA sequencing data analysis.

Supplemental Table S4. Primers used in this study.

ACKNOWLEDGMENTS

We thank André Specht, Hildegard Thölke, and Hartmut Wieland for tech-nical assistance, Xiaoye Liu for help with the statistical analyses, and NievesMedina Escobar for assistance with the preparation of embryos.

Received January 4, 2016; accepted March 31, 2016; published March 31, 2016.

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