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Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development Jing Xu1, Lin Zhang1, Dong-Lei Yang2, Qun Li1 and Zuhua He1 1

National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, Shanghai 200032, China; 2State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China

Summary Author for correspondence: Zuhua He Tel: +86 21 5492412 Email: [email protected] Received: 8 February 2015 Accepted: 23 May 2015

New Phytologist (2015) 208: 1089–1103 doi: 10.1111/nph.13530

Key words: Arabidopsis thaliana, conservation, pyrimidine, salvage pathway, thymidine kinase.


Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated.
Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development.
Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 50 -Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 20 -deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms.
Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development.

Introduction Nucleotide metabolism is critical for fundamental cellular and biochemical processes during organism growth and development. Nucleotides not only provide building blocks for DNA and RNA biosynthesis but also act as the energy carriers in metabolism. Nucleotides are among the components of essential coenzymes such as flavin adenine dinucleotide (FAD), S-adenosylmethionine (SAM) and nicotinamide adenine dinucleotide (NAD+) (Moffatt & Ashihara, 2002; Stasolla et al., 2003a; Zrenner et al., 2006, 2009). In addition, intermediates of nucleotide catabolism can be recycled into central metabolic pools of phosphate, nitrogen and carbon or become components of secondary metabolites such as caffeine and cytokinin (Mok & Mok, 2001; Smith & Atkins, 2002; Ashihara et al., 2008; Bar-Peled & O’Neill, 2011). Nucleotide synthesis in plants is similar to that in mammals and microorganisms, including de novo and salvage pathways (Nara et al., 2000; Smith & Atkins, 2002; Boldt & Zrenner, 2003; Kafer et al., 2004). The de novo synthesis of nucleotides results from the catalytic reaction of 5-phosphoribosyl-1-pyrophosphate (PRPP) with other small molecules such as carbon dioxide, amino acids and tetrahydrofolate, in which Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

ribonucleotide reductases catalyze the rate-limiting step (Wang & Liu, 2006; Garton et al., 2007). In the salvage pathway, nucleobases and nucleosides are phosphorylated to the corresponding nucleoside monophosphates by the activities of specific phosphoribosyltransferases and nucleoside kinases, respectively. It has been proposed that the salvage pathway is less energy consuming than the de novo synthesis pathway, because the synthesis of purine consumes five ATP molecules, whereas salvage requires only one (Moffatt et al., 2002). During different stages of plant growth and development, such as embryo maturation (Ashihara et al., 2001; Stasolla et al., 2001), seed germination (Stasolla et al., 2003b; Jung et al., 2009) and storage organ development (Geigenberger et al., 2005), the salvage pathway may provide an alternative source of nucleotides. The importance of the nucleotide salvage enzymes has been illustrated by molecular genetic analysis. Loss of function of the Arabidopsis thaliana adenine phosphoribosyltransferase (APT), which catalyzes the conversion from adenine to AMP, led to male sterility (Allen et al., 2002). Adenosine kinase (ADK)-deficient plants showed abnormal morphology, including malformed leaves and a dwarf and bushy stature, and reduced fertility. The failure to salvage adenosine also leads to a feedback inhibition of New Phytologist (2015) 208: 1089–1103 1089 www.newphytologist.com

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SAM-dependent transmethylation (Moffatt et al., 2002; Carrari et al., 2005). Of the six uracil phosphoribosyltransferases, enzymes that transfer phosphoribosyl from PRPP to uracil to form UMP, UPP has been identified to contribute almost all the UPRT activity, and UPP T-DNA mutants showed pleiotropic phenotypes including pale-green/albino leaves, dwarfism and sterility (Islam et al., 2007; Mainguet et al., 2009). Although no visible growth defect was found in single mutants of UKL1 or UKL2 (uridine kinase-like) their double mutant exhibited pale-green cotyledons at early growth stages and reduced biomass (Chen & Thelen, 2011). Therefore, the salvage pathway also plays important roles in maintaining appropriate growth and development in plants. Thymidine kinase (TK) is a key enzyme in the salvage synthesis of thymidine 50 -monophosphate (dTMP) from thymidine (dT). Intracellular dTMP is quickly phosphorylated to dTDP by thymidylate kinase (TMPK) and further to its triphosphate form by nucleoside diphosphate kinase (NDK) for DNA synthesis and repair. It has been reported that TK plays an important catalytic role in regulating the dNTP pool (Rampazzo et al., 2007). In human cells, two TKs, HsTK1 and HsTK2, have been identified. HsTK1 is located in the cytoplasm, and, as its activity is correlated with the proliferative activity of tumor cells, it is used as a marker in cancer clinics (Barth et al., 2008; Andrei et al., 2013). HsTK2 is located in the mitochondria and functions in a cellcycle-independent pattern. Mutations in HsTK2 are associated with the myopathic form of mitochondrial DNA depletion syndrome (MDS) caused by the shortage of mitochondrial dTTP (Pontarin et al., 2003; Wang et al., 2003). However, knowledge of TK functions in plants has been limited until recently. Clausen et al. (2012) demonstrated that A. thaliana extracts can catalyze all the deoxyribonucleosides including thymidine, and two TKs were identified according to sequence similarity and enzymatic activity analysis. They also found that TKs function redundantly during early organ development, as a single TK mutant grew normally. However, the detailed analysis of their functions is still obscure. In this study, we performed detailed analysis of the two TKs in A. thaliana, including analysis of their in vitro and in vivo enzymatic activities, expression patterns and subcellular localizations, and a comprehensive survey of mutant phenotypes throughout development. We also characterized the single-copy TK in rice (Oryza sativa) for the first time and found that both rice and human TKs complemented the defects of A. thaliana TK mutants, suggesting that TKs in different organisms shared conserved functions during evolution.

Materials and Methods Plant material and growth conditions Seeds of Arabidopsis thaliana (L.) Heynh (Columbia (Col-0) ecotype) carrying mutations in the genes At3g07800 (SALK_097767C, GK-401F10) and At5g23070 (SALK_074256C, GK-143D03) were obtained from the Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu/) and The European New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

Arabidopsis Stock Center (NASC, http://arabidopsis.info/), and these mutants were named tk1-1, tk1-2, tk2-1 and tk2-2 for convenience. Seeds of wild type (Col-0) and mutants were surface-sterilized and sown on half-strength Murashige and Skoog (MS) medium with 0.7% agar (Murashige & Skoog, 1962) and germinated in a controlled growth chamber (16 h : 8 h, day : night; 21°C) after cold treatment for 48 h at 4°C. At 6 d after germination (DAG), seedlings were transferred to soil. For the 50 -fluorodeoxyuridine (FdU; SigmaAldrich) toxicity experiments, FdU powder was dissolved in dimethylsulfoxide to produce a 0.1 M stock solution. Seeds from wild-type and homozygous mutant plants were germinated on half-strength MS solid medium with 300 nM or 1 lM FdU for 6 d. For feeding experiments, seeds were germinated and grown on half-strength MS solid medium supplemented with 20 -deoxyadenosine 50 -monophosphate (dAMP), dTMP, 20 -deoxyguanosine 50 -monophosphate (dGMP), 20 -deoxycytidine 50 -monophosphate (dCMP) and 20 -deoxyuridine 50 -monophosphate (dUMP). The feeding concentrations were 300 lM for both dTMP, dCMP and dUMP, 100 lM and 1 mM for both dAMP and dGMP. Genotyping and RNA analysis Genomic DNA was extracted for PCR from the mutant and wild-type plants as described by Edwards et al. (1991). The T-DNA insertions were detected with primer combinations of tk1-1RP/LBa1, tk1-2RP/3144, tk2-1RP/LBa1 and tk2-2RP/ 3144 for tk1-1, tk1-2, tk2-1 and tk2-2, respectively; tk1-1LP/ tk1-1RP and tk1-2LP/tk1-2RP for the wild-type sequence of tk1-1 and tk1-2, respectively; tk2-1LP/tk2-1RP for the wild-type sequence of tk2-1 and tk2-2 (Supporting Information Table S1). PCR amplifications were performed in a 20-ll reaction mixture including 100 ng of genomic DNA, 2 ll of 910 PCR buffer, 2 ll of 2.5 mM dNTPs, 1 ll each of 5 lM primers and 0.5 U Taq DNA polymerase (MBI Fermentas, Elyria, OH, USA). The thermal cycling consisted of initial denaturation at 95°C for 5 min, followed by 36 cycles of denaturation at 95°C for 30 s, annealing at a primer-specific temperature for 30 s and extension at 72°C for 1 min per kb, and a final extension at 72°C for 5 min. Homozygous T-DNA insertion mutants were crossed to produce F1 plants and then the derived F2 populations were genotyped to select double mutants. Total RNA was extracted from 2-wk-old seedlings using TRIzol reagent (Invitrogen) and treated with RNase-free DNase RQ1 (Promega). cDNA was synthesized from 3 lg of total RNA using an oligo (dT) primer and SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. RT-PCR analysis was performed using gene-specific primers, namely TK1F/TK1R for AtTK1, and TK2-1F/TK2-1R and TK2-2F/TK2-2R for AtTK2 (Table S1). Quantitative real-time PCR was carried out using SYBR premix Ex Taq (TaKaRa, Dalian, China) in a CFX96 Real-Time System (Bio-Rad). All the primers for target genes showed good linear amplification of cDNA by gradient dilution, and the Actin-2 gene was amplified as an internal control. Two-step PCR was performed by initial denaturation at 95°C for 15 s followed by 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 65°C for Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

New Phytologist 20 s. Data for each sample were obtained from three technical replicates and confirmed by the use of two independent biological samples. Expression ratios were calculated using the 2DDCt method. Histological detection of GUS fusion reporter expression The 1.4- and 3-kb promoter regions of AtTK1 (pTK1) and AtTK2 (pTK2) were amplified using primers pTK1F/pTK1R, and pTK2F/pTK2R with suitable enzymatic sites at their 50 ends (Table S1), respectively. The fragments were introduced into the binary vector pBI101 (GenBank accession number U12639.1) and the resulting GUS fusion reporter plasmids were transformed into Col-0 (Clough & Bent, 1998). Ten independent lines expressing pTK1::GUS and seven independent lines expressing pTK2::GUS were analyzed for GUS staining. Subcellular localization experiment AtTK1, AtTK2 and DAtTK2 cDNA was amplified by specific primers TK1-YF/TK1-YR, TK2-1-YF/TK2-YR, TK2-YF/TK2-GR and DTK2-YF/TK2-YR with suitable enzymatic sites at their 50 ends (Table S1), respectively, and then introduced in-frame into the plasmid pA7-GFP or pA7-YFP (kindly provided by Prof. Hongquan Yang) to produce fusion constructs of TK1-YFP, TK2-YFP, TK2-GFP and DTK2-YFP. The resulting fusions were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis thaliana mesophyll protoplasts isolated from 4-wk-old wild-type plants were transfected with different constructs following the procedure described by Yoo et al. (2007), and YFP alone was transfected as a control. To determine the mitochondrial location of TK2, the protoplasts were co-transfected with constructs of TK2-GFP and (alternative oxidase) AOX-RFP (Carrie et al., 2007). Protoplasts expressing the proteins were imaged under a laser scanning confocal microscope using a PlanApochromat 9100/1.4 oil objective (LSM510 META NLO; Zeiss). The fusion protein cassettes were also inserted into the binary vector 35S-pCambia1301 (GenBank accession number AF234297) to produce constructs 35S::AtTK1-YFP, 35S:: AtTK2-YFP and 35S::DAtTK2-YFP, which were transformed into Col-0 to generate stable transgenic plants. The fluorescence was measured on homozygous transgenic plants (T2 generation). Subcellular analysis of rice (Oryza sativa L.) TK followed a similar procedure. Moreover, the rice constructs and the HsTK1 construct were transformed into double mutants to determine their ability to recover the normal phenotype.

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from bacterial lysates by affinity column chromatography using glutathione-sepharose4B (GE Healthcare, Piscataway, NJ, USA), and then the protein concentration was measured using the Bradford assay. TK activity was determined by HPLC following the procedure described by Munch-Petersen et al. (1991) with slight modifications. The reaction mixture (1 ml) consisted of 50 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 2.5 mM ATP, 10 mM DTT, 0.5 mM cholamidopropyl dimethylammonio propanesulfonate (CHAPS), 3 mg ml1 bovine serum albumin (BSA), and 2 mM deoxynucleoside (dA, dG, dC, dT and dU). The reaction was initiated by the addition of purified recombinant proteins (5 mg ml1 protein) to the assay mixture at 37°C for 3 h, and terminated by heat incubation at 70°C for 10 min. The reaction mixture was cooled on ice for 30 min, and then centrifuged at 12 000 g for 10 min. The supernatants were filtered through a membrane filter (0.2 lm; Millipore) and analyzed by HPLC (Agilent 1260, Agilent Technologies, Santa Clara, CA, USA) using a ZORBAX SBC18 4.6 9 250 mm column (Agilent Technologies) for detection of dAMP, dTMP, dGMP and dUMP or a Paroshell ECC18 2.7 9 5 cm column (Agilent Technologies) for detection of dCMP. Functional complementation assay in E. coli The constructs pGEX-AtTK1, pGEX-AtTK2, pGEX-DAtTK2, pGEX-OsTK, pGEX-OsDTK and pGEX-HsTK1 were transformed into TK-deficient E. coli strain KY895 (F-, k-, tdk-1, IN (rrnD-rrnE)1, ilv-276) (Hiraga et al., 1967). Positive transformants were determined by PCR and subsequent sequencing (Table S1). Functional complementation of TK activity was determined on minimal medium M9 (0.6% Na2HPO4, 0.3% KH2PO4, 0.05% NaCl, 0.1% NH4Cl, 0.1 mM CaCl2, 1 mM MgSO4 and 0.2% glucose) supplied with either 1 lM 30 -azido20 ,30 -dideoxythymidine (AZT; Sigma-Aldrich) or 10 lM FdU. A pGEX empty vector was used as a negative control, and pGEXHsTK1 was used as a positive control. EdU incorporation assay Three-day-old seedlings were incubated in half-strength MS liquid medium with 10 lM 5-ethynyl 20 -deoxyuridine (EdU) for 45 min or 3 h, and then were transferred to the medium that contained the EdU-detection cocktail (C10350; Invitrogen Life Technologies) for 30 min. The EdU-labeled root cells were observed using a laser scanning confocal microscope.

Recombinant protein expression and enzyme activity assay AtTK1, AtTK2, DAtTK2, HsTK1, OsTK and DOsTK cDNA was amplified by PCR using the relevant primers with suitable enzymatic sites at their 50 ends (Table S1). The PCR fragments were cloned into the pGEX-6p-3 vector (Invitrogen) and then expressed in Escherichia coli BL21 DE3 cells. Protein expression was induced with 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 30°C for 8 h. Recombinant proteins were purified Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

Transmission electron microscopy Samples were infiltrated for 30 min with fixation buffer (2.5% (v/v) glutaraldehyde in phosphate buffer (pH 7.2)) under vacuum. After overnight treatment at 4°C, samples were post-fixed in 0.1 M cacodylate (pH 7.4) with 2% OsO4 at 4°C, then processed as previously described (Li et al., 2011) and viewed by electron microscopy (Hitachi Ltd, Tokyo, Japan). New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

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AtTK1 and AtTK2 are guided to the cytosol and mitochondria separately (Fig. 2), similar to human HsTK1 and HsTK2. Such organelle localization and functionalization were also suggested by a recent study in which TK activity was detected both in the cytosol and in the mitochondria but not in the chloroplast (Clausen et al., 2014). In the thymidine salvage pathway, the human and yeast TMPKs catalyze the next step transforming dTMP to dTDP (Jong & Campbell, 1984; Su & Sclafani, 1991). AtTMPK in A. thaliana performs a similar function and encodes two isoforms by alternative splicing, one in the cytosol and another in the mitochondria (Ronceret et al., 2008). Therefore, dTMP produced by AtTK1 and AtTK2 would be separately phosphorylated to dTDP by the TMPK isoforms in the cytosol and mitochondria. On the basis of these results, we propose that AtTK1 and AtTK2 function through a coordination of expression patterns and subcellular localizations, to meet the nucleotide demands of the plant cell at different stages of development. AtTK1 and AtTK2 share the same catalytic properties in pyrimidine salvage The two TKs exhibited differential expression patterns and cellular locations, but their substrate specificities were similar, as revealed by direct measurement of enzyme activity (Fig. 3). In the human cell, HsTK1 phosphorylates only thymidine and dU, while HsTK2 phosphorylates all three pyrimidine nucleosides, including dC. Our results showed that both AtTK1 and AtTK2 can catalyze phosphorylation of dU, dT and dC, with low activity with dC. According to the previous report, the single deoxynucleotide kinase AtdNK had a broad substrate specificity for dA, dG, dC and dU but not thymidine (Clausen et al., 2012). However, dTMP depletion and thus dTTP insufficiency might be major consequences of TK deficiency, given that dUMP has no functional role in DNA duplication or mRNA transcription and can be converted to dTMP by thymidylate synthase (Balestrazzi et al., 1995; Neuburger et al., 1996). In addition, the disruption of dCMP supply is weak as both AtTKs have low activity to produce it, and dCMP can also be converted to dUMP by dCMP deaminase and then to dTTP (Reichard, 1988; Sanchez et al., 2012). We also found that a mutation in the rice dCMP deaminase gene led to a virescent leaf defect like that of tk mutants, suggesting that dTTP biosynthesis is important for chloroplast development (Xu et al., 2014). Consistently, the tk mutants could be recovered by any single pyrimidine monophosphate of dTMP, dUMP and dCMP. Therefore, it will be worth further dissecting the roles of these conversion enzymes in plant growth and development. Nucleotide salvage mediated by two TKs is critical to chloroplast development Interestingly, no visible defect could be found for the overexpression line tk1-2 or the knockout line tk1-1, while both the tk2 mutant lines showed slight growth defects (Fig. 5c–h), which was not described in previous report (Clausen et al., 2012). As the two TKs had similar catalytic properties, the different mutant New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

New Phytologist phenotypes are mostly attributable to their different expression patterns and subcellular locations. AtTK1 is highly expressed in proliferating regions, where the de novo synthesis of nucleotides is highly required. Therefore, the loss of AtTK1 function could be compensated by the de novo pathway. By contrast, the expression of AtTK2 is more widespread and localized to the mitochondria, suggesting that it may participate mainly in nucleotide salvage for mitochondrial DNA replication. It is interesting that mutants of AtTK1 and AtTK2 showed different reactions to the FdU treatment, with tk1-1 more resistant than tk2-1 and tk2-2 (Fig. 6c–e), which is likely to be attributable to their different subcellular localizations. The double mutants showed more severe developmental abnormalities (Fig. 5c–h), suggesting an interaction between the two TKs in different organelles. It has been demonstrated that dTTP could be transported between cytosol and mitochondria in the human cell (Pontarin et al., 2003), which could also be the case in plants. Previous studies suggested that the dNTP balance between the chloroplast, mitochondrion and nucleus is crucial for plant development, and under limited dNTP supply, organelle development is strongly inhibited (Zhao et al., 2000; Wang & Liu, 2006). We suggested that the AtTK2 mutation abolished the salvage of dTTP in mitochondria, but, as the critical energy organelles, mitochondria probably competed for the cytosolic dTTP pool, reducing the cytosolic dTTP supply to the chloroplasts. Indeed, one copy of AtTK1 (TK1/tk1-1tk2-1) is enough for the tk1-1tk2-1 mutant plants to survive, indicating that the depletion of dTTP by AtTK2 in the mitochondria should be compensated by the cytosol pyrimidine pool generated by AtTK1. Studies of the rice virescent 3 and strip1 mutants also suggested that plastid DNA synthesis for chloroplast biogenesis is less critical for plant survival and can be sacrificed under insufficient dNTP levels (Yoo et al., 2009). Therefore, the disruption of dTTP in tk mutants mostly inhibits plastid DNA synthesis and later chloroplast biogenesis. However, the mechanism by which the dNTP pool prioritizes nuclear and possibly mitochondrial DNA synthesis remains elusive. We propose that there exists a complicated mechanism or network to distribute dNTPs generated by the salvage and de novo pathways. TKs in different organisms are functionally conserved in salvaging nucleotides It has been recognized that several enzymes catalyzing the de novo pathway are evolutionarily conserved in all species. As an energysaving pathway, the salvage pathway can reuse preformed nucleotides to complement the de novo pathway. The A. thaliana TKs share conserved domains with the TKs found in E. coli (Hiraga et al., 1967) and in humans (Flemington et al., 1987; Wang et al., 1999, 2003), implying that they could have similar roles in pyrimidine nucleotide salvage. We transformed the AtTKs into an E. coli TK-negative strain, and found that the transformants could not survive on M9 medium supplied with AZT or FdU (Fig. 4), confirming their functional conservation. More importantly, we confirmed for the first time the conserved function of the rice and human TKs in A. thaliana through genetic Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

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Fig. 2 Subcellular localization of Arabidopsis thaliana THYMIDINE KINASE 1 (AtTK1) and AtTK2. (a) Transient expression of TK1-YFP, TK2-YFP, DTK2YFP and YFP in A. thaliana protoplasts, indicating that TK1 is localized to the cytosol, while TK2 showed a punctate localization pattern in the cytoplasm. The truncated TK2 protein without the transit peptide is localized to the cytosol as is TK1. Signals from YFP fluorescence (left panels), chlorophyll autofluorescence (middle panels) and merged images (right panels) are shown. (b) Co-localization of TK2-GFP with AOX-RFP, a mitochondrion-located maker, indicating that TK2 is localized to the mitochondria in A. thaliana protoplasts. (c) Fluorescence signals in 35S::AtTK1-YFP transgenic lines can be detected in the mesophyll cells, hypocotyl and epidermal hairs (from left to right are fluorescence signals). (d) Fluorescence signals in 35S::AtTK2-YFP transgenic lines can be detected in the mesophyll cells, root and epidermal hairs (from left to right). The red signal in mesophyll cells represents chlorophyll autofluorescence, which does not coincide with the AtTK2-YFP signal. (e) Fluorescence signals in 35S::DAtTK2-YFP transgenic lines can be detected in the mesophyll cells, hypocotyl and epidermal hairs (from left to right). Bars, 20 lm.

without the transit peptide) were produced for fluorescence visualization (Fig. 2a). The TK1-YFP signal could be detected both in the cytoplasm and in the nucleoplasm. However, TK2-YFP showed a punctate pattern in the cytoplasm which was distinct from the red chlorophyll autofluorescence. When the transit peptide was excluded (DTK2-YFP), the punctate pattern of TK2-YFP disappeared and TK2-YFP became ubiquitous, like the YFP control, indicating that the transit peptide determined the cellular location of TK2. Furthermore, we observed that AtTK2-GFP was co-localized with the mitochondrial marker AOX-RFP (Fig. 2b), demonstrating that AtTK2 is localized to the mitochondria. To further confirm the subcellular localization of AtTK1 and AtTK2 revealed by transient expression, we developed stable transgenic plants (35S::AtTK1-YFP, 35S:: AtTK2-YFP and 35S::DAtTK2-YFP) in the Col-0 background. YFP signals were detected in mesophyll cells, epidermal hairs, the hypocotyl and the root (Fig. 2c–e). The protein location patterns were consistent with the observations in the protoplast. Therefore, the subcellular localizations are different for the two A. Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

thaliana TKs, with AtTK1 in the cytosol and AtTK2 in the mitochondria, suggesting a functional compartmentation. AtTK1 and AtTK2 have similar thymidine kinase activities As AtTK1 and AtTK2 displayed different expression patterns and subcellular localizations, whether they have the same or different thymidine kinase activity was investigated. The full-length coding sequences of AtTK1, AtTK2 and DAtTK2 were cloned into the expression vector pGEX-6p-3 to produce fusion proteins with glutathione S-transferase (GST) in E. coli, which were purified by affinity chromatography (Fig. S2). The purified proteins were incubated with different nucleoside substrates and subsequently analyzed for kinase activity by HPLC following a procedure modified from the method reported by Munch-Petersen et al. (1991). The HPLC results showed that AtTK1, AtTK2 and DAtTK2 displayed the same substrate specificity in phosphorylating dT and dU to form dTMP and dUMP, respectively (Fig. 3c, d). Neither dA nor dG was converted by the TKs in this assay New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

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Fig. 3 Enzymatic activity of Arabidopsis thaliana THYMIDINE KINASE 1 (AtTK1) and AtTK2 in vitro. The distribution of substrates and products in the enzymatic analysis is shown in the chromatograms. The colored lines show different proteins added as follows: yellow, GST; pink, AtTK1; green, AtTK2; red, DAtTK2. Blue lines denote the positions of all the substrates and products in the reaction buffer. (a) AtTK1, AtTK2 and DAtTK2 cannot catalyze dA conversion. The red arrow indicates the position of the product dAMP not detected in the analysis. (b) AtTK1, AtTK2 and DAtTK2 cannot catalyze dG conversion. The red arrow indicates the position of the product dGMP not detected in the analysis. (c) AtTK1, AtTK2 and DAtTK2 displayed the same substrate specificity in phosphorylating dT to dTMP. The detectable products and substrates are highlighted by the red box. (d) AtTK1, AtTK2 and DAtTK2 displayed the same substrate specificity in phosphorylating dU to dUMP. The detectable products and substrates are highlighted by the red box. (e, f) AtTK1, AtTK2 and DAtTK2 displayed the same substrate specificity in phosphorylating dC to dCMP (f is a close-up view of e). The detectable products and substrates are highlighted by the red box.

(Fig. 3a,b). We also detected minor specificity for dC, as a tiny product peak was detected at the dCMP location on the chromatogram, suggesting a possible low deoxycytidine kinase activity (Fig. 3e,f). All these results establish that both AtTK1 and AtTK2 have thymidine kinase activity towards multiple pyrimidine nucleosides. AtTK1 and AtTK2 can complement a TK-deficient E. coli strain To demonstrate that AtTK1 and AtTK2 have TK activity in vivo, we performed a functional complementation assay New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

utilizing the E. coli system. AZT and FdU can be phosphorylated to AZT-MP and FdUMP, respectively, by the activity of EcTK, which are then quickly phosphorylated to AZT-TP and FdUTP by specific or ubiquitous nucleotide kinases. AZT-TP is a known telomerase inhibitor, blocking the elongation of the DNA chain (Liu et al., 2007). FdUTP acts as a mutagen and a potent inhibitor of thymidylate synthase, leading to the depletion of thymidine precursors in cellular pools (Khan et al., 2010). Therefore, the application of AZT and FdU can inhibit E. coli growth, and TK-deficient E. coli mutant strain KY895 can survive in the presence of AZT or FdU (Wang et al., 2000). The three TK constructs (pGEX-AtTK1, AtTK2 and DAtTK2) were Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

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Fig. 4 Functional complementation of thymidine kinase (TK)-deficient Escherichia coli by Arabidopsis thaliana, rice and human TKs. The sketch map shows the distribution of transformants with different TK genes. The pGEX empty vector was used as a negative control. Growth of transformants on M9 medium with or without 1 lM 30 -azido-20 ,30 -dideoxythymidine (AZT) or 10 lM 50 -fluorodeoxyuridine (FdU) is shown.

transformed into KY895 and cultured on M9 medium supplemented with AZT or FdU. Human thymidine kinase (HsTK1) was also transformed as a positive control. The results clearly show that the growth of the transformants expressing AtTK1, AtTK2 and DAtTK2 was greatly inhibited on the medium with 1 lM AZT or 10 lM FdU, similar to the positive HsTK1 control (Fig. 4). Therefore, the failure of strains expressing A. thaliana TKs to survive suggested that AtTK1 and AtTK2 have TK bioactivities that are similar to that of EcTK. AtTK1 and AtTK2 function together to sustain plant growth and chloroplast development To dissect the function of AtTK1 and AtTK2 in plant development genetically, we examined different T-DNA insertion mutants of AtTK1 and AtTK2 available from the A. thaliana community, including SALK_097767C (tk1-1), GK-401F10 (tk1-2), SALK_074256C (tk2-1) and GK-143D03 (tk2-2). As shown in Fig. 5(a), the insertion occurred either within the exon (tk1-1, tk2-1 and tk2-2) or in the 50 untranslated region (UTR) (tk1-2). A semiquantitative RT-PCR assay showed that tk1-1 was a null mutant, whereas tk1-2 exhibited increased expression of AtTK1 compared with wild-type Col-0 (Figs 5b, S3). We thus speculated that tk1-1 is a knockout mutant and tk1-2 is a line overexpressing AtTK1, as full-length transcripts can be amplified from homozygous tk1-2 plants. For AtTK2, two pairs of primers were used for RT-PCR analysis (Fig. 5a). No expression was detected in either tk2-1 or tk2-2 using TK2-1 primers, while higher expression was detected in tk2-2 compared with wild-type Col-0 when using TK2-2 primers that locate upstream of the insertion (Fig. 5b). Therefore, tk2-2 may produce a truncated TK2 protein. To determine whether these insertion mutations affected plant growth and development, homozygous plants were selected for observation. We did not observe obvious growth defects in either tk1-1 or tk1-2 mutant plants, while the tk2-1 and tk2-2 mutants had smaller yellow cotyledons under normal growth conditions (Fig. 5c,d). As the stable transgenic AtTK2overexpression lines grew normally (data not shown) and the virescent phenotype was more severe in tk2-1 than in tk2-2, we Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

considered tk2-2 as a weak mutant. To clarify the genetic interaction of the two TK genes, we developed tk1tk2 double mutants. The double mutant tk1-1tk2-1 showed an albino phenotype and died at an early stage (< 10 d old), whereas the heterozygous mutant TK1/tk1-1tk2-1 could survive to maturity, although with severely bleached leaves (Fig. 5c–g). The bleached leaves of TK1/ tk1-1tk2-1 became green as the plants grew, but their flowering was delayed (Fig. 5h, insert). Interestingly, the double mutant tk1-1tk2-2 survived and, like TK1/tk1-1tk2-1, developed yellow cotyledons and severely virescent leaves and showed delayed flowering (Fig. 5c–h). The differential morphology among the mutants suggested that AtTK1 and AtTK2 might function coordinately, and loss of both TKs greatly impaired plant vigor. To investigate cellular processes involved in the virescent leaf phenotype, the ultrastructure of chloroplast thylakoid membranes both in virescent and in normal seedlings (10 d old) was clarified by transmission electron microscopy (TEM). In Col-0 seedling leaves, chloroplasts were crescent-shaped and contained a well-formed thylakoid structure including stroma thylakoids and grana lamellae (Fig. S4a). By contrast, some chloroplasts in virescent leaves of TK1/tk1-1tk2-1 and tk1-1tk2-2 lacked thylakoid membranes (Fig. S4b,c), some formed rudimentary thylakoids consisting of only granal stacks but no stroma lamellae (Fig. S4d,e), and some developed lamellar structures with normally stacked grana and stroma lamellae similar to the widetype chloroplasts (Fig. S4f). We did not find obvious changes in the mitochondria of the mutants (data not shown), despite the fact that AtTK2 is localized to the mitochondria. FdU resistance and EdU incorporation confirm loss of TK activity in mutants As FdU can block DNA biosynthesis and thereby inhibits growth of E. coli and A. thaliana (Wu & King, 1994), the effect of FdU on growth of the single and double mutants was tested (Fig. 6a–e). We observed that tk1-1 and tk1-1tk2-2 exhibited increased resistance to FdU (300 nM) in comparison with Col-0 (Fig. 6c), and tk1-1tk2-2 was more resistant to FdU (1 lM) than tk1-1 (Fig. 6d). The higher FdU resistance suggests a greater New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

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Fig. 5 Phenotypes of Arabidopsis thaliana mutants with different T-DNA insertions in THYMIDINE KINASE (AtTK) genes. (a) Schematic representation of annotated AtTK1 and AtTK2 and T-DNA insertion locations in the mutants. Triangles indicate the positions of T-DNA insertions; black and white boxes represent exons and untranslated regions (UTRs), respectively. A flag with a name indicates the primer position for RT-PCR analysis. (b) Transcription levels of AtTK1 and AtTK2 in Columbia (Col-0) and the mutants revealed by RT-PCR. Actin2 was amplified as a control. (c–h) Phenotypes of single and double mutants in sequential developmental stages. Plants grown on half-strength Murashige and Skoog (MS) medium were photographed at 3 d after germination (DAG) (c; bars, 1 mm), 6 DAG (d; bars, 2 mm) and 10 DAG (e; bars, 4 mm). After germination on half-strength MS medium for 6 d, seedlings were transferred into the soil and photographed at 15 d (f; bar, 1.2 cm), 30 d (g; bar, 1.2 cm) and 36 d (h; bar, 4 cm). Inserts in (h) show the magnified leaves from the TK1/tk1-1tk2-1 and tk1-1tk2-2 plants.

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Fig. 6 Reduced sensitivity of the Arabidopsis thaliana thymidine kinase (tk) mutants to cytotoxins. (a) Schematic diagram showing the distribution of mutants on the plate. (b) Plants grown on half-strength Murashige and Skoog (MS) medium without 50 -fluorodeoxyuridine (FdU). (c) Plants grown on half-strength MS medium supplemented with 300 nM FdU, indicating that tk1-1tk2-2 and tk1-1 had reduced FdU sensitivity. Note that only tk1-1 and tk1-1tk2-2 and the segregated tk1-1tk2-1 seedlings from TK1/tk1-1tk2-1, and not the other seedlings, could grow on the medium. (d) Plants grown on half-strength MS medium supplemented with 1 lM FdU. Note that the tk1-1tk2-2 seedlings still grew under high concentrations of FdU. (e) Close-up of the plants grown in (d) to highlight the progeny of TK1/tk1-1tk2-1. Arrows indicate the tk1-1tk2-1 plants with elongated hypocotyls. (f) Fluorescence detection in roots of 3-d-old Columbia (Col-0) and mutant seedlings incubated with EdU for 45 min. (g) Fluorescence signals from Col-0, tk1-1tk2-1 and tk1-1tk2-2 roots incubated with EdU for 3 h. Note that the mutations in the TK genes greatly decreased the EdU-triggered fluorescence. Control check (CK) was a negative control, in which the Col-0 seedlings were incubated in half-strength MS liquid medium without EdU, and then transferred to the EdU-detection cocktail. Bars: (f, g) 40 lm.

depletion of thymidine kinase activity in mutant plants. We further confirmed that all surviving seedlings separated from the offspring of TK1/tk1-1tk2-1 under 1 lM FdU were tk1-1tk2-1 (Fig. 6e). Taken together, the results indicated reduced TK activity in the mutants. To further confirm reduced catalytic activity in these tk mutants in vivo, we performed the EdU incorporation assay in the root. EdU is a terminal alkyne-containing nucleoside analog of thymidine. TK can phosphorylate EdU to form EdUMP and finally EdUTP, which is incorporated into DNA (Kotogany et al., 2010). The coupled reaction could generate fluorescence in the presence of Alexa Fluor® dye containing the azide groups (Invitrogen Life Technologies). The results showed that there were no EdU-labeled cells either in tk1-1tk2-1 or in tk1-1tk2-2 after incubation for 45 min (Fig. 6f). When the incorporation time was extended to 3 h, weak fluorescence signals could be found in tk1-1tk2-2 but still not in tk1-1tk2-1 (Fig. 6g). The fluorescence in the single mutants indicated that each of the TKs Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

alone has catalytic activity in vivo. Therefore, the results confirmed that TK activity is greatly reduced or completely lost in the double mutants, disrupting the salvage of pyrimidine nucleotides for normal plant growth and development. External feeding of dTMP, dUMP and dCMP alleviates mutant defects We have demonstrated that the two TKs can use the same substrates and catalyze the conserved salvage reaction, but their intrinsic effects on recycling nucleotides needed to be further determined. To address this, we fed the single and double mutants with nucleoside monophosphates (dAMP, dGMP, dTMP, dUMP and dCMP). dAMP and dGMP had no effect on either single mutants or double mutants (Fig. S5), confirming that purine nucleosides were not used as a substrate by the two TKs. When grown on a medium supplemented with 300 lM dTMP, dUMP or dCMP, the tk2-1, tk2-2 and tk1-1tk2-2 New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

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Fig. 7 Recovery of the Arabidopsis thaliana mutant plants by feeding with dTMP, dUMP, dCMP and a mixture. Seeds were germinated on half-strength Murashige and Skoog (MS) supplemented with 300 lM dTMP, dUMP and dUMP and a mixture as indicated. Photographs of 6-d-old seedlings are shown. The results indicated that tk2-1, tk2-2 and tk1-1tk2-2 developed normal green leaves like Columbia (Col-0), while TK1-1/tk1-1tk2-1 and tk1-1tk2-1 only partially recovered with pale/variegated cotyledons. Bars, 2 mm.

mutant plants exhibited green leaves. Moreover, the nucleotide feeding allowed the severe mutant TK1/tk1-1tk2-1 to recover almost completely and even the lethal mutant tk1-1tk2-1 could survive and produce green first true leaves (Figs 7, S6). We also measured the chlorophyll content of mutants feeding with different pyrimidine nucleotides, and the results were consistent with the visual observations (Fig. S6f). These results indicate that loss of TK activity reduced the production of pyrimidine nucleotides, and disturbed the chloroplast development and plant growth of A. thaliana.

plants survived and developed variegated cotyledons and true leaves, although many of them were dwarf in comparison with Col-0 (Fig. 8a,b). Similarly, OsTK could also functionally complement E. coli strain KY895 (Fig. 4). Subcellular location analysis showed that the OsTK-GFP fusion protein was localized to the mitochondria (Fig. 8c–f), similar to that of AtTK2. Moreover, like OsTK, HsTK1 could also recover the albino phenotype of tk1-1tk2-1 (Fig. 8g). Therefore, our study indicated that the function of TKs is conserved during evolution of diverse organisms.

Rice and human TKs share a conserved function with AtTK1 and AtTK2

Discussion

We have shown that the A. thaliana TKs could complement the E. coli mutant strain, as did the human TK. In order to determine whether TKs from other organisms have a conserved function in salvaging nucleotides in A. thaliana, we performed genetic complementation experiments with the rice and human TKs in the tk1-1tk2-1 mutant. The rice genome has only one TK gene (OsTK; LOC_Os03g02200) with an N-terminal transit peptide like AtTK2 (Fig. S1). An enzymatic assay indicated that OsTK and N-terminal truncated ΔOsTK could phosphorylate thymidine, dU and dC to dTMP, dUMP and dCMP, reminiscent of the effect of AtTK1 and AtTK2 (Fig. S7). When OsTK was constitutively expressed in the lethal tk1-1tk2-1 mutant, the New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

Functions of AtTK1 and AtTK2 are coordinated Thymidine kinase is a crucial enzyme in the salvage pathway of nucleotide metabolism, which recycles the preformed nucleotides to meet the demand of DNA synthesis and repair. In this study, the molecular characteristics of AtTK1 and AtTK2 were studied comprehensively. The two genes appear to have coordinating functions in plant development, as AtTK1 is mainly expressed in highly proliferating cells (Fig. 1a), while AtTK2 displays more widespread expression, in both young and mature tissues (Fig. 1b). This resembles the expression profile of the human TK genes, with HsTK1 highly expressed in dividing S-phase cells, while HsTK2 is highly expressed in resting cells (P
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Fig. 8 Functional complementation of Oryza sativa THYMIDINE KINASE (OsTK) and Homo sapiens TK (HsTK) in Arabidopsis thaliana. (a, b) Phenotypes of albino-lethal tk1-1tk2-1 and the representative transgenic lines tk1-1tk2-1/OsTK (T4, T6 and T10). The OsTK complemented plants exhibited green true leaves at the seedling stage (a; bars, 1 mm) and finally grew to adult plants (b; bars, 2 cm). (c–e) Subcellular localization of OsTK in 35S::OsTK-GFP transgenic A. thaliana. Fluorescence signal was detected in epidermal hairs (c), hypocotyl cells (d) and root cells (e). Bar, 20 lm. (f) Co-localization of OsTK-GFP with AOX-RFP in A. thaliana protoplasts. Bar, 30 lm. (g) Rescue of lethal tk1-1tk2-1 mutant plants by overexpression of HsTK1. T2, T3 and T5 represent three independent transgenic lines, which could grow to adult plants. Bar, 2 cm.

et al., 2008). The E2F cis-element is involved in the transition from G1 to S phase of plant cells (Chaboute et al., 2002; Stevens et al., 2002; Vandepoele et al., 2005), and such elements are also present in the HsTK1 promoter (Tommasi & Pfeifer, 1997). Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

Interestingly, we found a putative E2F-binding site in the AtTK1 promoter. Moreover, AtTK1 is up-regulated by E2F-DP overexpression (Vlieghe et al., 2003), suggesting that AtTK1 expression relies on cell cycle control. New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

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AtTK1 and AtTK2 are guided to the cytosol and mitochondria separately (Fig. 2), similar to human HsTK1 and HsTK2. Such organelle localization and functionalization were also suggested by a recent study in which TK activity was detected both in the cytosol and in the mitochondria but not in the chloroplast (Clausen et al., 2014). In the thymidine salvage pathway, the human and yeast TMPKs catalyze the next step transforming dTMP to dTDP (Jong & Campbell, 1984; Su & Sclafani, 1991). AtTMPK in A. thaliana performs a similar function and encodes two isoforms by alternative splicing, one in the cytosol and another in the mitochondria (Ronceret et al., 2008). Therefore, dTMP produced by AtTK1 and AtTK2 would be separately phosphorylated to dTDP by the TMPK isoforms in the cytosol and mitochondria. On the basis of these results, we propose that AtTK1 and AtTK2 function through a coordination of expression patterns and subcellular localizations, to meet the nucleotide demands of the plant cell at different stages of development. AtTK1 and AtTK2 share the same catalytic properties in pyrimidine salvage The two TKs exhibited differential expression patterns and cellular locations, but their substrate specificities were similar, as revealed by direct measurement of enzyme activity (Fig. 3). In the human cell, HsTK1 phosphorylates only thymidine and dU, while HsTK2 phosphorylates all three pyrimidine nucleosides, including dC. Our results showed that both AtTK1 and AtTK2 can catalyze phosphorylation of dU, dT and dC, with low activity with dC. According to the previous report, the single deoxynucleotide kinase AtdNK had a broad substrate specificity for dA, dG, dC and dU but not thymidine (Clausen et al., 2012). However, dTMP depletion and thus dTTP insufficiency might be major consequences of TK deficiency, given that dUMP has no functional role in DNA duplication or mRNA transcription and can be converted to dTMP by thymidylate synthase (Balestrazzi et al., 1995; Neuburger et al., 1996). In addition, the disruption of dCMP supply is weak as both AtTKs have low activity to produce it, and dCMP can also be converted to dUMP by dCMP deaminase and then to dTTP (Reichard, 1988; Sanchez et al., 2012). We also found that a mutation in the rice dCMP deaminase gene led to a virescent leaf defect like that of tk mutants, suggesting that dTTP biosynthesis is important for chloroplast development (Xu et al., 2014). Consistently, the tk mutants could be recovered by any single pyrimidine monophosphate of dTMP, dUMP and dCMP. Therefore, it will be worth further dissecting the roles of these conversion enzymes in plant growth and development. Nucleotide salvage mediated by two TKs is critical to chloroplast development Interestingly, no visible defect could be found for the overexpression line tk1-2 or the knockout line tk1-1, while both the tk2 mutant lines showed slight growth defects (Fig. 5c–h), which was not described in previous report (Clausen et al., 2012). As the two TKs had similar catalytic properties, the different mutant New Phytologist (2015) 208: 1089–1103 www.newphytologist.com

New Phytologist phenotypes are mostly attributable to their different expression patterns and subcellular locations. AtTK1 is highly expressed in proliferating regions, where the de novo synthesis of nucleotides is highly required. Therefore, the loss of AtTK1 function could be compensated by the de novo pathway. By contrast, the expression of AtTK2 is more widespread and localized to the mitochondria, suggesting that it may participate mainly in nucleotide salvage for mitochondrial DNA replication. It is interesting that mutants of AtTK1 and AtTK2 showed different reactions to the FdU treatment, with tk1-1 more resistant than tk2-1 and tk2-2 (Fig. 6c–e), which is likely to be attributable to their different subcellular localizations. The double mutants showed more severe developmental abnormalities (Fig. 5c–h), suggesting an interaction between the two TKs in different organelles. It has been demonstrated that dTTP could be transported between cytosol and mitochondria in the human cell (Pontarin et al., 2003), which could also be the case in plants. Previous studies suggested that the dNTP balance between the chloroplast, mitochondrion and nucleus is crucial for plant development, and under limited dNTP supply, organelle development is strongly inhibited (Zhao et al., 2000; Wang & Liu, 2006). We suggested that the AtTK2 mutation abolished the salvage of dTTP in mitochondria, but, as the critical energy organelles, mitochondria probably competed for the cytosolic dTTP pool, reducing the cytosolic dTTP supply to the chloroplasts. Indeed, one copy of AtTK1 (TK1/tk1-1tk2-1) is enough for the tk1-1tk2-1 mutant plants to survive, indicating that the depletion of dTTP by AtTK2 in the mitochondria should be compensated by the cytosol pyrimidine pool generated by AtTK1. Studies of the rice virescent 3 and strip1 mutants also suggested that plastid DNA synthesis for chloroplast biogenesis is less critical for plant survival and can be sacrificed under insufficient dNTP levels (Yoo et al., 2009). Therefore, the disruption of dTTP in tk mutants mostly inhibits plastid DNA synthesis and later chloroplast biogenesis. However, the mechanism by which the dNTP pool prioritizes nuclear and possibly mitochondrial DNA synthesis remains elusive. We propose that there exists a complicated mechanism or network to distribute dNTPs generated by the salvage and de novo pathways. TKs in different organisms are functionally conserved in salvaging nucleotides It has been recognized that several enzymes catalyzing the de novo pathway are evolutionarily conserved in all species. As an energysaving pathway, the salvage pathway can reuse preformed nucleotides to complement the de novo pathway. The A. thaliana TKs share conserved domains with the TKs found in E. coli (Hiraga et al., 1967) and in humans (Flemington et al., 1987; Wang et al., 1999, 2003), implying that they could have similar roles in pyrimidine nucleotide salvage. We transformed the AtTKs into an E. coli TK-negative strain, and found that the transformants could not survive on M9 medium supplied with AZT or FdU (Fig. 4), confirming their functional conservation. More importantly, we confirmed for the first time the conserved function of the rice and human TKs in A. thaliana through genetic Ó 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist Ó 2015 New Phytologist Trust

New Phytologist complementation (Fig. 8). However, the transgenic plants did not fully recover; which could be explained by the dosage effect of transgenes with different expression levels in our transgenic experiment. Another possibility is that the rice and human TKs might not fully perform enzymatic functions in A. thaliana cells, with subtle evolutionary variations in these functions. Nevertheless, the current study clearly demonstrates that TKs have conserved activity in the salvage pathway from lower to higher organisms. Intriguingly, the rice genome probably has only one TK that is localized to the mitochondria, in contrast to A. thaliana. According to the genome annotation in Gramene (http://www.gramene.org/), maize (Zea mays) and sorghum (Sorghum bicolor) also have only one TK. However, two dNKs were predicted in rice and maize genomes, but only one in A. thaliana. The difference in copy numbers of TKs and dNKs may reflect the adaptive selection of gene duplication after the divergence of monocotyledons and dicotyledons, and the loss of a single-copy gene might be compensated by other genes with similar functions. The role of the single OsTK should be further investigated in future studies to help us understand nucleotide salvage in monocotyledons and its differences from that in dicotyledons. It will also be necessary to study the mechanism of nucleotide distribution between chloroplasts, mitochondria and the nucleus.

Acknowledgements We thank Jiqin Li and Xiaoshu Gao for technical assistance and Genyun Chen for performing protein purification. We acknowledge Hongquan Yang (Shanghai Jiao Tong University) for providing the vector pA7-YFP/ pA7-GFP and James Whelan (University of Western Australia) for providing the AOX-RFP plasmid. This work was supported by a grant from the National Natural Science Foundation of China (91117018).

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Sequence alignment of TK proteins. Fig. S2 The purified GST and GST fusion proteins GSTAtTK1, GST-AtTK2, GST-DAtTK2, GST-OsTK and GSTDOsTK. Fig. S3 Higher expression level of AtTK1 was detected in tk1-2 than in Col-0.

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Fig. S4 Ultrastructure of chloroplasts in mesophyll cells of 10-dold Col-0 (WT) and the virescent mutants.

Fig. S7 Enzymatic activity of OsTK and DOsTK toward dT, dU and dC.

Fig. S5 Non-rescue of Arabidopsis thaliana mutant phenotypes by feeding dAMP and dGMP.

Table S1 Primers used in this study

Fig. S6 Morphology and chlorophyll contents of thymidine kinase (tk) mutants feeding with dTMP, dUMP, dCMP and their mixture.

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