Triphosphate Tunnel Metalloenzyme Function inSenescence Highlights a Biological Diversification ofThis Protein Superfamily1[OPEN]

Huoi Ung,a,2 Purva Karia,a,2 Kazuo Ebine,c,d Takashi Ueda,c,d Keiko Yoshioka,a,b,3 and Wolfgang Moedera,3

aDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3B2, CanadabCenter for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, ON M5S 3B2,CanadacDivision of Cellular Dynamics, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi444-8585, JapandDepartment of Basic Biology, Graduate University for Advanced Studies, Okazaki, Aichi 444-8585, Japan

ORCID IDs: 0000-0003-3889-6183 (W.M.); 0000-0002-3797-4277 (K.Y.).

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphatesubstrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsisthaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved inpathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of thebiological roles between AtTTM1 and 2. Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similarto AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutantanalysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionallyup-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. Thedouble mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biologicalfunction. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopyrevealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transientoverexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, weshow that two TTMs display the same biochemical properties but distinct biological functions that are governed by theirtranscriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death andsenescence through novel mitochondrial tail-anchored proteins.

The triphosphate tunnel metalloenzyme (TTM) su-perfamily comprises two groups of enzymes, RNAtriphosphatases and CYTH phosphatases (CyaB aden-ylate cyclase, thiamine triphosphatase) that possess

common characteristics in their catalytic sites (Iyer andAravind, 2002; Gong et al., 2006). Members of this su-perfamily are able to hydrolyze a variety of triphos-phate substrates, giving them important roles in cAMPformation, mRNA capping, and secondary metabolism(Iyer and Aravind, 2002; Gallagher et al., 2006; Gonget al., 2006; Song et al., 2008). Most TTMs possess aunique tunnel structure composed of eight antiparallelbeta strands forming a beta barrel and a characteristicEXEXK motif (where X is any amino acid), which isimportant for catalytic activity (Lima et al., 1999; Iyerand Aravind, 2002; Gallagher et al., 2006). In addition,TTMs also share the requirement of a divalent metalcation cofactor, usually Mg2+ or Mn2+ (Bettendorff andWins, 2013).

While the catalytic activity of some TTMs has beenelucidated, the biological function of most TTM pro-teins is unknown. However, it appears they have ac-quired the ability to act on a diverse range of nucleotideand organophosphate substrates (Iyer and Aravind,2002; Bettendorff and Wins, 2013). Known functions ofTTMs include fungal and protozoan RNA triphospha-tases (Cet1; Lima et al., 1999; Gong et al., 2006), bacterial

1 This article was supported by a Discovery Grant from the Natu-ral Science and Engineering Research Council of Canada (grant no.PGPIN-2014-04114), Canadian Foundation for Innovation, and On-tario Research Fund to K.Y., grants from the Ministry of Education,Culture, Sports, Science and Technology of Japan (24114003 and15H04382) to T.U., and a graduate student fellowship from the On-tario government to H.U.

2 These authors contributed equally to the article.3 Address correspondence to wolfgang.moeder@utoronto.ca and

keiko.yoshioka@utoronto.ca.The 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:Keiko Yoshioka (keiko.yoshioka@utoronto.ca).

W.M, H.U., and K.Y. designed the research; H.U., P.K., K.E., andT.U. performed the research; H.U., P.K., W.M., and K.Y. analyzed thedata; W.M., H.U., and K.Y. wrote the article.

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adenylate cyclases (CyaB from Aeromonas hydrophilaand YpAC-IV from Yersinia pestis; Sismeiro et al., 1998;Gallagher et al., 2006), and mammalian thiamine tri-phosphatases (Lakaye et al., 2004; Song et al., 2008).More recently, tripolyphosphatase activity was dis-covered for CthTTM from Clostridium thermocellum andNeuTTM from Nitrosomonas europaea, highlighting thefunctional diversity of this superfamily (Keppetipolaet al., 2007; Delvaux et al., 2011). In some instances,TTM proteins can also possess additional domains,adding further complexity to their range of functions.

Plant genomes contain two types of TTMs: one with asingular CYTH domain and one with a CYTH domainfused to a P-loop kinase domain (Iyer and Aravind,2002). In Arabidopsis (Arabidopsis thaliana), there arethree TTM family members, AtTTM1, 2, and 3:AtTTM1and 2 belong to the latter type, possessing a uridinekinase (UK) domain in addition to the CYTH domain,while AtTTM3 belongs to the former type, possessingonly a singular CYTH domain (Supplemental Fig. S1).Previously, we demonstrated that AtTTM3 exhibitstripolyphosphatase activity and may play a role in rootdevelopment (Moeder et al., 2013), whereas AtTTM2displays pyrophosphatase (PPase) activity and is anegative regulator of pathogen defense responses (Unget al., 2014). Interestingly, publicly available microarraydata show significantly different expression patternsfor AtTTM1 and AtTTM2. This suggests that whileAtTTM1 and AtTTM2 possess the same domain ar-rangement, these genes may play distinct roles whereAtTTM1 may be involved in leaf senescence.

Leaf senescence is an active and highly regulatedprocess where nutrients are remobilized to othergrowing tissues of the plant. Individual cells within aleaf undergo metabolic changes in order to dismantleeach component before programmed cell death (PCD)occurs and sink-source relationships begin to transition.The initiation of leaf senescence naturally occurs byaging but can also be induced by a range of externalfactors such as drought, darkness, and hormones(abscisic acid, ethylene), resulting in the visible loss ofchlorophyll or yellowing, since the chloroplasts are thefirst organelles to be disassembled (Weaver et al., 1998;Breeze et al., 2011). Leaf senescence is generally be-lieved to be a special form of PCD, which shares some,but not all, of the characteristics of PCD (van Doorn andWoltering, 2004).

Usually a carefully orchestrated dismantling of thechloroplast is one of the first events in the senescenceprocess (Keech et al., 2007). Other organelles, such asmitochondria and peroxisomes, remain active muchlonger and provide energy for the cell as well as takeover a number of metabolic tasks during this process(Keech et al., 2007; Chrobok et al., 2016). While yeastand animals possess mitochondrial PPases (Lundinet al., 1991; Curbo et al., 2006), so far no mitochondrialPPase has been identified in plants. But generallyPPases remove pyrophosphate (PPi) that is created as abyproduct of anabolic processes such as nucleic acid,protein, and carbohydrate synthesis (Gómez-García

et al., 2006). Removal of PPi is necessary to prevent theinhibition of thermodynamically unfavorable reactions(Maeshima 2000). Another function could be to keepcytosolic [PPi] levels low, since high [PPi] is toxic(Cooperman et al., 1992).

Here, we show that AtTTM1 is localized to the mi-tochondrial outer membrane and, just like its closeparalog AtTTM2, displays in vitro PPase activity.AtTTM1 is a positive regulator of dark-induced andnatural leaf senescence. It shares high sequence simi-larity with its paralog, AtTTM2, and AtTTM2 canfunctionally complement the knockout phenotype ofAtTTM1. Moreover,AtTTM2 overexpression canmimicAtTTM1’s cell death-inducing function. In spite of theirsimilarities, these two genes do not appear to be in-volved in the same biological processes. Rather, it istheir different gene expression patterns that dictatetheir distinct biological roles.

RESULTS

AtTTM1 and AtTTM2 Share Many Common Properties

Arabidopsis possesses three genes that are annotatedas CYTH domain/TTM proteins. AtTTM3 comprisesonly a CYTH domain, while the CYTH domain followsan N-terminal uridine kinase domain in AtTTM1 andAtTTM2. AtTTM1 and 2 also possess a coiled-coil do-main and a transmembrane domain at the C-terminalend (Supplemental Fig. S1A). AtTTM1 and 2 share highamino acid sequence similarity with over 92% sequencesimilarity and approximately 66% sequence identity,which is even higher in the UK (81% identity) andCYTH domains (73% identity; Supplemental Fig. S1B).This suggests that AtTTM1 and 2 have either identicalor very similar enzymatic properties. Most other dicotplant species also encode TTM1 and TTM2 orthologs,which fall into two distinct clades, indicating conserveddistinct functions for AtTTM1 and 2 (Supplemental Fig.S1C).

Enzymatic Properties of AtTTM1 and AtTTM2

Plant CYTH domain proteins have been annotated asadenylate cyclases based on their sequence similarityto the adenylate cyclase from Aeromonas hydrophila(Sismeiro et al., 1998; Iyer and Aravind, 2002). How-ever, all three recombinantly expressed TTM proteinsfrom Arabidopsis did not display adenylate cyclaseactivity (Moeder et al., 2013; Ung et al., 2014). Rather,we previously demonstrated that AtTTM3 displaysstrong tripolyphosphatase activity with a weaker af-finity for nucleotide triphosphates (Moeder et al., 2013).On the other hand, AtTTM2 showed a strong prefer-ence for pyrophosphate (PPi; Ung et al., 2014). AtTTM1revealed the same properties as AtTTM2, with highestactivity for PPi andweaker activities for ATP, ADP, andtripolyphosphate (PPPi; Fig. 1A). Further biochemicalanalyses revealed a slight preference for alkaline pH

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(pH 8–9) aswell as a strong cofactor preference forMg2+

(Fig. 1, B and C). The Km for pyrophosphate was de-termined to be 16.7 6 5.2 mM (Vmax = 284 6 19 nmolmin21 mg21) and 17 6 3.2 mM (Vmax = 366 6 15 nmol

min21 mg21) for AtTTM1 and AtTTM2, respectively(Fig. 1, D and E).

The N-terminal kinase domain is a typical P-loopkinase, which was annotated as a uridine/cytidine ki-nase. It has conserved Walker A, Walker B, and lidmodulemotifs (Supplemental Fig. S2; Leipe et al., 2003).Thus, the uridine kinase activity of recombinant pro-teins was tested. Both AtTTM1 and 2 did not displayactivity on a uridine substrate, while a known Arabi-dopsis uridine kinase (AtUKL1; Islam et al., 2007) pro-duced uridine monophosphate as expected (SupplementalFig. S3). This suggests that the kinase domain acts on adifferent substrate than uridine.

AtTTM1 and AtTTM2 Are Differentially Expressed acrossVarious Tissues

Since AtTTM1 and AtTTM2 both displayed similarcatalytic activities, it raised the question whether theyare redundant or whether they have taken on differentbiological roles. Thus, the expression patterns of AtTTM1and AtTTM2 were examined using the Bio-Analytic Re-source (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi;Schmid et al., 2005; Winter et al., 2007). While AtTTM2was predominantly expressed in the shoot apices ofinflorescences, AtTTM1 appeared to be ubiquitouslyexpressed in all tissues,with strong expression detected insenescent leaves (Supplemental Fig. S4). This suggeststhat while the same catalytic activity was detected forboth AtTTM1 and AtTTM2, the biological roles they playmay be different.

ttm1 Plants Do Not Show Altered Disease Resistance

The differences in the expression patterns of AtTTM1and 2 prompted us to further analyze the differences intheir transcriptional regulation. It has been shown thatAtTTM2 is a negative regulator of the SA-dependentamplification loop for pathogen defense responses andis also transcriptionally suppressed upon pathogen in-fection and treatments with the pathogen-associatedmolecular pattern, flg22 (flagellin peptide), the defensehormone salicylic acid (SA) or the biological analog ofSA, benzothiadiazole (BTH; Ung et al., 2014). Therefore,we monitored the transcript levels of AtTTM1 after in-fection with the avirulent Hyaloperonospora arabidopsidis(Hpa) isolate, Emwa1. In contrast to AtTTM2, AtTTM1expression was not down-regulated upon pathogen in-fection while the Pathogenesis-related1 (PR1) gene, amarker of pathogen resistance activation, was stronglyinduced, as expected (Fig. 2A; Ung et al., 2014), indi-cating a fundamental difference in the transcriptionalregulation between AtTTM1 and 2. This result suggeststhat AtTTM1 does not play the same role as AtTTM2 inpathogen defense.

Next, two allelic T-DNA insertion knockout lines ofAtTTM1, ttm1-1 and ttm1-2, were isolated for pheno-typic characterization. Both mutants exhibited no dis-cernible morphological difference to Columbia wild

Figure 1. Both AtTTM1 and AtTTM2 display pyrophosphatase activity.A, Substrate specificity of AtTTM1 and AtTTM2. Enzymatic activity wastestedwith various phosphate compounds. Reactionswith PPi, ATP, andPPPi were performed at pH 9 in the presence of 0.5 mM substrate,2.5 mM Mg2+ cofactor, and 2 mg of protein. Reactions with ADP wereperformed in the same conditions except with 0.03 mM substrate toreduce background phosphate readings. Glutathione S-transferase(GST) served as a negative control. Each bar represents the mean 6 SE

(n = 3). The experiment was repeatedmore than three times with similarresults. B, Pyrophosphatase activity as a function of pH in the presenceof 0.5 mM PPi, 2.5 mM Mg2+, and 2 mg of protein. Each data point rep-resents the mean6 SE (n = 3). The experiment was repeated twice withsimilar results. C, Divalent cation cofactor specificity of pyrophospha-tase activity. Reactions were performed at pH 9 in the presence of0.5 mM PPi, 2.5 mM cation cofactor, and 2 mg of protein. Each barrepresents the mean 6 SE (n = 3). The experiment was repeated twicewith similar results. (D and E) Pyrophosphatase activity as a function ofpyrophosphate concentration. Reactions were performed at pH 9 in thepresence of 0 to 600 mM PPi, 2.5 mM Mg2+, and 2 mg of AtTTM1 (D) andAtTTM2 (E) protein. Each data point represents the mean 6 SE (n = 3).The experiment was repeated twice with similar results.

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type (Col; Supplemental Fig. S5). Following infectionwith the avirulent Hpa isolate, Emwa1, as reportedprior, ttm2 mutants developed significantly more hy-persensitive response (HR) cell death compared to wildtype, indicating enhanced defense activation (Fig. 2, BandC;Ung et al., 2014). On the other hand ttm1mutantsdisplayed a subtle but statistically significant attenua-tion of HR cell death, but no effect on fungal growthwas observed. Furthermore, infection with the virulentHpa isolate, Emco5, revealed that ttm2 plants displayedsignificantly less growth of the pathogen, while ttm1mutants showed no significant difference from wild-type plants (Fig. 2, D and E). These data suggest thatAtTTM1 does not play the same role as AtTTM2 inpathogen defense.

ttm1 Displays Delayed Senescence Phenotypes

Since transcriptional regulation of AtTTM2 is tightlyconnected to its biological role (Ung et al., 2014), we in-vestigated the transcriptional regulation of AtTTM1 inorder to gain insight into its biological role. A noticeableincrease in AtTTM1 transcript levels was observed insenescing leaves (Supplemental Fig. S4). Furthermore,several coexpressed genes were senescence-associatedgenes, such as a caspase-like protease, gamma vacuo-lar processing enzyme (g-VPE), receptor-like proteinkinase1, SENESCENCE-ASSOCIATED GENE SAG12,and a Cys proteinase (Supplemental Table S1; Expres-sion Angler, http://bar.utoronto.ca/ntools/cgi-bin/ntools_expression_angler.cgi; Toufighi et al., 2005).Therefore, we hypothesized that AtTTM1 is involved inleaf senescence and assessed transcriptional changesduring senescence using the well-established dark-induced senescence assay (Riefler et al., 2006). We firstvalidated the microarray data by monitoring the ex-pression levels of AtTTM1 in detached leaves over thecourse of 7 d in darkness. As shown in Figure 3,AtTTM1transcript levels were already over 3-fold increased after1 d and continued to increase until 7 d in darkness,

Figure 2. ttm1 does not show enhanced immunity to Hyaloper-onospora arabidopsidis. A, AtTTM1 expression is not suppressed bypathogen infection. Quantitative real-time PCR analysis of AtTTM1(left) or PR1 (right, as a control of infection) expression in Hyaloper-onospora arabidopsidis (Hpa) isolate, Emwa1-infected (Emwa1), orwater-treated (H2O) cotyledons 7 d after infection withHpa. Transcriptswere normalized to AtEF1a. Shown are the averages of three indepen-dent experiments. Each bar represents the mean6 SE (n = 3). An asteriskindicates significant difference to the H2O control, (Student’s t test, p,0.001). All samples represent a pool of 14 to 20 seedlings. B Infectionphenotype of Columbia wild type (Col), ttm1-1, and ttm2-1 mutantplants 10 d after infection with the avirulent Hpa isolate, Emwa1.Shown is Trypan blue staining of infected cotyledons (Cot) revealing HR

cell death (white arrows) and some hyphae (red arrows in Col and ttm1)and uninfected first true leaves (TL) revealing enhanced HR in ttm2plants. Scale bar = 250 mm. C, HR index of Hpa isolate Emwa1 infec-tion. Stained leaves were microscopically examined and assigned todifferent classes (see right; n = 45). The experiment was repeated threetimes with similar results. tm2-1 displayed significantly more HR thanCol (Fisher’s exact test, P = 0), while ttm1-1 showed less HR than Colwild type (P = 0.005). D, Infection phenotype of Col wild type, ttm1-1,and ttm2-1mutant plants 12 d after infection with virulent Hpa, isolateEmco5. Shown is Trypan blue staining of infected cotyledons reveal-ing hyphae (white arrows) and oospores (red arrows) in wild type andttm1-1 and reduced hyphal growth in ttm2-1. Scale bar = 250 mm. E,Disease index of Hpa isolate Emco5 infection. Stained leaves were mi-croscopically examined and assigned to different classes (right; n = 30). Asignificant difference was detected between ttm2-1 and Col (Fisher’sexact test, P , 0.0001), but not between ttm1-1 and Col. Highly sig-nificant differences are marked by an asterisk. The experiment was re-peated four times with similar results.

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whereas AtTTM1 levels rose to over 8-fold. On the otherhand, AtTTM2 transcript levels showed only a mild in-crease after 1 d but did not increase further over thecourse of the experiment (Fig. 3).Next, we monitored the dark-induced senescence

phenotype in ttm1 mutants. Leaves 3 to 6 of 4- to5-week-old plants were detached and floated on waterin the dark. Strikingly, they consistently displayed lesschlorophyll loss during the course of the experiment(Fig. 4A). This difference was observed as early as 5 dafter dark treatment and was most dramatic 7 d after.Measurement of the total chlorophyll content con-firmed this observation quantitatively (Fig. 4B). Toconfirm that the difference in chlorophyll retention wasindeed due to the absence of AtTTM1, a genomic frag-ment comprising the promoter region and the AtTTM1gene was introduced into the ttm1 knockout mutantbackground, and two independent transgenic lineswere analyzed. Complementation of the ttm1 mutantplants with wild-type AtTTM1 rescued the delayedsenescence phenotype of ttm1, returning chlorophyllretention to wild-type levels (Fig. 4, C and D).To correlate the loss of chlorophyll in ttm1 mutants

with a senescence response, the expression of severalsenescence markers were monitored. CAB6 and SAG13are known to be down-regulated and up-regulated, re-spectively, during the transition from vegetative growthto senescence, whereas SAG12 is senescence specific andis strongly induced when this process is activated(Lohman et al., 1994). In ttm1 mutant leaves, the tran-scriptional down-regulation ofCAB6was delayed by 2 dcompared towild type over the course of 7 d in darkness(Fig. 4E). In addition, SAG13 transcript levels were visi-bly lower in ttm1mutant leaves compared to wild type.Furthermore, SAG12 expression was starkly induced at5 d after darkness inwild-type leaves but did not expressin ttm1mutant leaves until 7 d (Fig. 4E). Taken together,these data suggest thatAtTTM1 is a positive regulator ofdark-induced senescence.

To investigate whether ttm1 mutants also displaydelayed senescence in intact plants, we first placedwhole plants in the dark and assess the chlorophyllcontent after 5 d. Similar to our detached leaf assay, wealso observed less chlorophyll loss in intact ttm1mutantplants (Supplemental Fig. S6). To further test whetherAtTTM1 also plays a role in natural senescence, plantswere grown under 16 h light and monitored for firstsigns of leaf yellowing. At 5 weeks, wild-type plantsstarted to display first signs of yellowing on the oldestleaves, while comparable leaves of ttm1 plants remainedgreen (Fig. 5A). The ttm1/TTM1 complementation plantsshowed the same timing of yellowing aswild-type plants.The chlorophyll content of these leaves was also signifi-cantly higher in ttm1 plants compared to the wild typeand ttm1/TTM1 complementation plants (Fig. 5B).

ttm2 Does Not Show a Senescence Phenotype, and the ttm1ttm2 Double Mutant Does Not Exhibit Additive Effects

Transcriptional analysis of AtTTM2 upon dark-induced senescence treatment suggested that AtTTM2is not involved in senescence and that its biological roleis different from that of AtTTM1. To further validatethis point, we first analyzed the dark-induced senes-cence phenotype of ttm2mutants. As shown in Figure 6,ttm2 plants showed the same degree of chlorophyll lossas wild-type leaves. Combined with the absence of adisease-resistance phenotype in ttm1 plants, this indi-cates thatAtTTM1 and 2 are not biologically redundant.To further address this point and also investigate pos-sible synergistic effects, double-knockout lines weregenerated by cross pollination, and their phenotypeswere compared to their respective single-knockoutlines. The ttm1 ttm2 double mutant did not displayany morphological differences compared to wild type(Fig. 6A). After 7 d in the dark, the ttm1 ttm2 doublemutant displayed the same chlorophyll retention as thettm1 single mutant, while the ttm2 single mutant be-haved like wild type, suggesting that only AtTTM1, butnotAtTTM2, plays a role in senescence (Fig. 6, B and C).

Pathogen infection using the virulent Hpa isolate,Emco5, was also performed on ttm1, ttm2, and the ttm1ttm2 double mutant. The double mutant displayed aphenotype similar to the ttm2 single mutant, while ttm1plants behaved like Col wild type plants (Fig. 6, D andE). These data suggest that AtTTM1 is involved in se-nescence, whereas AtTTM2 plays a role in pathogendefense, further indicating their involvement in inde-pendent biological processes.

AtTTM1 Is Localized to the MitochondrialOuter Membrane

To determine the subcellular localization of AtTTM1,ttm1 plants were transformedwith a pTTM1:YFP-TTM1construct. To confirm that the fusion protein is prop-erly localized and functional, several independentlines were assessed in the dark-induced senescence

Figure 3. AtTTM1 is upregulated in senescent leaves. Quantitative real-time PCR analysis of AtTTM1 and AtTTM2 expression in detached leavesof 4- to 5-week-old Arabidopsis accession Columbia (Col) wild-typeplants after dark treatment. Transcripts were normalized to AtEF1a. Eachbar represents the mean 6 SE (n = 3). Bars marked with different lettersindicate a significant difference to their own 0 time point (P , 0.05).

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complementation assay (Supplemental Fig. S7A). Threeindependent lines displayed the same complementa-tion phenotype as the complementation line withoutYFP (Fig. 4D). Four- to ten-day-old seedlings were thenused for confocal microscopy. YFP-AtTTM1 displayeda punctate pattern in two independent transgenic lines(Fig. 7; Supplemental Fig. S7B). In order to determinewhich subcellular compartment these puncta represent,we first analyzed the effect of wortmannin and bre-feldin A. The YFP pattern was not affected by these in-hibitors, indicating that AtTTM1 is not localized to theGolgi, trans-Golgi network, or endosome (SupplementalFig. S8). On the other hand, the YFP pattern clearly colo-calized with the MitoTracker dye (Fig. 7; SupplementalFig. S7), suggesting a mitochondrial localization. Further-more, higher magnification images showed that the YFPsignal surrounds the MitoTracker signal, indicating a lo-calization in the mitochondrial outer membrane (Fig. 7B).Indeed, AtTTM1 has a transmembrane domain at its Cterminus (Fig. 7C) and has been predicted to be a tail-anchored protein that is localized to mitochondria(Kriechbaumer et al., 2009). A pTTM1:YFP-TTM1 DTMconstruct completely lost the mitochondrial localizationand instead was found in the cytosol (Supplemental Fig.S7B). This construct also failed to complement the ttm1senescence phenotype (Supplemental Fig. S7A), suggest-ing that the mitochondrial localization is required for itsbiological function.

On the other hand, a YFP signal from plants transformedwith the pTTM2:YFP-TTM2 construct couldnot bedetected,most likely due to the low expression levels of AtTTM2(Supplemental Fig. S4). Therefore, YFP-taggedAtTTM1 and2 under the CaMV35S promoter were transiently expressedin N. benthamiana. In both cases a similar punctate patternwas observed for both AtTTM1 and 2 (Supplemental Fig.S9). This and the fact that Kriechbaumer et al. (2009) andMarty et al. (2014) also identified AtTTM2 as a tail-anchorprotein suggests that AtTTM2 is also localized to the mito-chondrial outer membrane.

AtTTM1 and AtTTM2 Can Functionally ComplementEach Other

AtTTM1 and AtTTM2 exhibit distinct expressionpatterns and knockout phenotypes. Furthermore, thedouble mutant did not display enhanced phenotypesfor either pathogen resistance or delayed senescence

Figure 4. ttm1 displays delayed dark-induced leaf senescence. A,Leaves of 5-week-old Arabidopsis accession Columbia (Col) wild typeand ttm1 mutants were detached and floated for 7 d on water in thedark. Pictures were taken at day 0 and day 7. Scale bar = 1cm. B, Totalchlorophyll content was measured 0 and 7 d after dark treatment. Eachbar represents the mean 6 SE (n = 3). An asterisk denotes significance

difference toCol wild type (P, 0.01). The experimentwas repeated threetimes with similar results. C, Leaves of 5-week-old Col wild type, ttm1-1,and two independent ttm1/TTM1 complementation lines were detachedand floated for 7 d on water in the dark. Pictures were taken at day 0 andday 7. Scale bar = 1cm. D, Total chlorophyll content was measured 0 and7 d after dark treatment. Each bar represents the mean 6 SE (n = 3). Barsmarked with different letters indicate a significant difference to Col wildtype (P , 0.0001). The experiment was repeated twice with similar re-sults. E, Expression of senescence markers CAB6, SAG12, and SAG13 indetached leaves of Col wild type and ttm1-1 mutant plants after darktreatment. b-tubulin (TUB) served as loading control.

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(Fig. 6A). However, the two genes have very high se-quence similarity, all known catalytic amino acid resi-dues are conserved between them (Supplemental Fig.S2), and their subcellular localization is most likely thesame. Thus, we asked whether the biological functionof the two genes is solely conferred by their expressionpatterns. To address this question, functional comple-mentation was conducted using four promoter swapconstructs: (1) theAtTTM1 promoter followed by eitherthe coding sequence (CDS) of AtTTM1 or AtTTM2 and(2) the AtTTM2 promoter followed by the CDS of eitherAtTTM1 or AtTTM2. Three independent transgeniclines each were analyzed. The AtTTM1 promoter lineswere subjected to the dark-induced senescence assay,revealing that both the AtTTM1 as well as the AtTTM2CDS can complement the chlorophyll retention

Figure 5. The ttm1 mutant displays delayed natural senescence. A,Natural senescence phenotype of 5-week-old Col wild type, ttm1, andttm1/TTM1 complementation plants. At this time, the oldest leaves ofCol plants start to yellow (white arrows), which is not seen in ttm1plants. B, Total chlorophyll of the oldest leaves (leaves 1–3) of the aboveplants. Each bar represents the mean 6 SE (n = 3). Bars marked withdifferent letters indicate a significant difference (P , 0.05). The exper-iment was repeated three times with similar results.

Figure 6. The ttm1 ttm2 double mutant does not exhibit additive effectson senescence and pathogen resistance phenotypes. A, Morphologicalphenotype of Arabidopsis accession Columbia (Col) wild type, ttm1-1,ttm1-2, and ttm1-1 ttm2-1 plants. Photos showapproximately 5-week-oldplants. Scale bar = 1 cm. B, Leaves of 5-week-old Col wild type, ttm1-1,ttm1-2, and ttm1-1 ttm2-1 plants were detached and floated on water inthe dark. Pictures were taken at day 0 and day 7. Scale bar = 500 mm. C,Total chlorophyll content was measured at 0 and 7 d after dark treatment.Each bar represents the mean 6 SE (n = 3). Bars marked with differentletters indicate a significant difference (P , 0.01). The experiment wasrepeated three times with similar results. D, Infection phenotype of Colwild type, ttm1-1, ttm1-2, and ttm1-1 ttm2-1 plants 11 d after infectionwith the virulent Hpa isolate, Emco5. Shown is Trypan blue staining ofinfected cotyledons revealing hyphae (white arrows) and oospores (redarrows). Scale bar = 250 mm. E, Stained leaves were microscopicallyexamined and assigned to different classes (see below; n= 105–112, fromfour independent experiments). Fisher’s exact test was performed. Sig-nificant differences are marked by an asterisk.

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phenotype of ttm1 (Fig. 8A). To confirm that the pro-moter constructs behaved as expected, quantitativereal-time PCR was used to analyze the up-regulation ofboth AtTTM1 and AtTTM2 under the control of theAtTTM1 promoter after 7 d of dark treatment (Fig. 8B).In the reverse experiment, the AtTTM2 promoter lineswere subjected to Hpa infection. Quantitative real-timePCR analysis confirmed that both AtTTM1 andAtTTM2 under the control of the AtTTM2 promoterwere similarly down-regulated after treatment withBTH, as previously shown (Supplemental Fig. S10A;Ung et al., 2014). As shown in Supplemental FigureS10B, only one of three pTTM2:TTM2 and two out ofthree pTTM2:TTM1 independent lines showed a clearrescue of the enhanced disease resistance phenotype ofttm2 (Supplemental Figure S10B).

These data indicate the possibility of functionalsimilarity of these proteins. However, since comple-mentation of ttm2 was not seen in all transgenic lines,we further tested transient overexpression of bothgenes under the control of the CaMV35S promoter inN. benthamiana. We found that transient overexpressionof AtTTM1 induced senescence-like cell death 7 d afterAgrobacterium infiltration when the plants were keptin the dark (Fig. 8C). However, this cell death wasnot observed when plants were kept in the light (Fig.8D), which matches AtTTM1’s function during dark-induced senescence. As suggested by the complementation

analysis, overexpression of AtTTM2 also induced celldeath in the dark (Fig. 8, C and D). Taken together,these data indicate that the in planta enzymatic functionof AtTTM1 and AtTTM2 is identical or at least verysimilar. This suggests that the different expressionpattern of these two genes is critical for their specificbiological functions.

DISCUSSION

The TTM superfamily is characterized by an activesite situated within a tunnel comprised of antiparallelb-sheets. Furthermore, members of this superfamily acton triphosphate substrates with a strict dependency ona metal cation cofactor. TTM proteins are present in allliving organisms, where they have taken up a range ofdifferent functions. What they have in common isthat their substrates contain triphosphate moieties(Bettendorff and Wins, 2013). Plants are unique in twoways: First, they possess usually three TTM genes,while most other organisms only encode one type (Iyerand Aravind, 2002; Bettendorff and Wins, 2013). Sec-ond, AtTTM3 and its orthologs are comprised of only aCYTH domain whileAtTTM1 and 2 and their orthologsdisplay an additional N-terminal uridine kinasedomain. This fusion of a uridine kinase and a CYTHdomain is only seen in plants and members of theslime mold family (Mycetozoa), such as Dictyosteliumdiscoideum (Iyer and Aravind, 2002). Interestingly, D.discoideum also encodes two TTM genes, udkC and udkD(uridine-cytidine kinase; http://dictybase.org/).Another striking feature of this group is that the twoglutamates of the EXEXK motif are not conserved(TYILK in AtTTM1 and 2, IYILK and VYVCK in udkCand D, respectively), as are a number of the conservedbasic and acidic residues facing into the b-barrel. In-terestingly, the K of the EXEXK domain still is con-served, and all four proteins have a P residue before themodified EXEXK domain (other TTM proteins typicallyhave an aliphatic residue in that position—I, L, or V).

We have previously characterized AtTTM3 as a tri-polyphosphatase with a potential function in root de-velopment (Moeder et al., 2013) and AtTTM2 as anegative regulator of the SA amplification loop duringpathogen resistance responses (Ung et al., 2014).Knockout lines of AtTTM1, which shows 65% identityand 92% similarity to AtTTM2 at the amino acid level,did not display enhanced disease resistance, but rathershowed a delayed senescence phenotype, as was ex-pected based on our analysis of coexpressed genes.When senescence was induced by dark treatment ofdetached leaves, ttm1 plants retained chlorophyll lon-ger than wild-type plants. Furthermore, they displayeddelayed induction of the senescence marker genes,SAG12 and SAG13, suggesting that AtTTM1 plays arole during the senescence process.

The fact that the ttm1 delayed senescence phenotypewas also observed after whole-plant dark treatmentand during natural senescence clearly indicates that

Figure 7. YFP-TTM1 localizes to the mitochondria. A, Ten-day-oldseedlings of pTTM1:YFP-TTM1 plants were analyzed by confocal mi-croscopy. The YFP signal localized around the mitochondria stainedwith 50 nM MitoTracker Orange. Scale bar = 10 mm. B, Higher mag-nification observation of the mitochondria stained with MitoTrackerOrange, which are surrounded by the YFP signal, indicating a locali-zation of AtTTM1 in themitochondrial outermembrane. Scale bar = 10mm.C, Domain structure of AtTTM1. (UK = uridine kinase, CYTH = CyaB thia-mine triphosphatase domain, CC = coiled-coil domain, TM = transmem-branedomain). Bottom, Sequence of the transmembrane domain ofAtTTM1and AtTTM2 (black) and the C-terminal tail (red).

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AtTTM1 is an important (integral) part of the senes-cence pathway. Further evidence for this comes fromseveral transcriptome studies (Lin and Wu, 2004;Buchanan-Wollaston et al., 2005). Buchanan-Wollastonet al. (2005) compared the transcriptional response ofnatural senescence with dark-induced senescence. Theyfound that 827 genes were upregulated in senescentleaves compared to nonsenescent leaves. Of thesegenes, approximately 53% were also upregulated indark-induced senescent leaves. Many of these geneseither have putative or determined roles in macromol-ecule degradation, carbohydrate metabolism, mem-brane transport, secondary metabolism, and autophagy.This suggests that there are clear molecular differencesbetween natural and dark-induced senescence; however,the overlapping genes may constitute a core senes-cence pathway of components required for the executionof senescence (van der Graaff et al., 2006). AtTTM1 isupregulated in both data sets, andwe observed a delayedonset of natural senescence in the ttm1mutant, indicatingthat it likely plays a central role in the senescence pro-gram. Interestingly, AtTTM1 was also upregulated inanother data set, which identified genes that are inducedduring starvation-induced senescence of suspensioncells (Swidzinski et al., 2002). These cells also exhibitedclear symptoms of PCD. AtTTM1 is part of a group of229 genes that are upregulated in all three types of se-nescence, suggesting that AtTTM1 is part of a core senes-cence pathway of components required for the executionof senescence (van der Graaff et al., 2006).

Here, we connect both AtTTM1 and AtTTM2 to PCDduring senescence and pathogen-induced HR, respec-tively. Their precise molecular role(s) during theseprocesses are not clear yet, but their mitochondrial lo-calization is likely crucial for their biological function,since TTM1 DTM, which does not localize to the mito-chondria, lost its ability to complement the ttm1mutantphenotype. The involvement of mitochondria in plantPCD has been suggested but is not understood well(Lam et al., 2001; Qamar et al., 2015; Li et al., 2016).Based on the presence of the C-terminal transmem-brane domains (Fig. 7C; Kriechbaumer et al., 2009) andour confocal analyses, we conclude that AtTTM1 and2 are mitochondria-localized, tail-anchored proteins.These proteins are usually localized in the mitochondrial

Figure 8. AtTTM1 and AtTTM2 can complement each other. A, Totalchlorophyll content was measured in Arabidopsis accession Columbia(Col) wild type, ttm1-1, and three independent ttm1-1 complementa-tion lines expressing AtTTM1 under its native promoter (pTTM1:TTM1)or AtTTM2 under the TTM1 promoter (pTTM1:TTM2) 0 and 7 d afterdark treatment of detached leaves. Each bar represents the mean 6 SE

(n = 3). Bars marked with different letters indicate a significant differ-ence (P , 0.05). The experiment was repeated four times with similarresults. B, Quantitative real-time PCR analysis of AtTTM1 and AtTTM2expression in detached leaves of 4- to 5-week-old transgenic plants0 and 7 d after dark treatment. Transcripts were normalized to AtEF1a.Each bar represents the mean 6 SE (n = 3). An asterisk indicates signif-icant differences to day 0, Student’s t test, p, 0.01). C and D, Transientoverexpression of CaMV35S:YFP-AtTTM1 and CaMV35S:YFP-AtTTM2induces cell death in N. benthamiana 7 d after infiltration of A. tume-faciens when plants are kept in the dark (C), but not in the light (D).TEV = negative control (TEV HcPro). Red circles indicate cell death;white circles indicate no cell death.

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outer membrane with their catalytic domain facing to-ward the cytosol (Abell and Mullen, 2011). We could notunequivocally confirm the mitochondrial localization ofAtTTM2 at the same resolution as AtTTM1 due to lowexpression levels of AtTTM2 (under its native promoter),but the facts that (1) it was also predicted to be a tail-anchored protein (Marty et al., 2014; Fig. 7C) and (2)our analysis of transiently expressed AtTTM2 (under thestrongCaMV35S promoter) showed an identical punctatepattern (Supplemental Fig. S9) strongly suggest thatAtTTM2 is also a mitochondrial protein as AtTTM1.Furthermore, the fact that overexpression of eitherAtTTM1 or 2 induced senescence-like cell death in thedark supports our conclusion that AtTTM1 is involved insenescence-associated cell death and both proteins havethe same or very similar enzymatic activity.

A possible function of AtTTM1 could be in pyrimi-dine catabolism through its uridine kinase-like domain.Stasolla et al. (2004) suggested that decreased salvage ofuracil and uridine and increased salvage of thymidinerepresent a metabolic switch for the induction of pro-grammed cell death (PCD). However, recombinantlyexpressed AtTTM1 (and 2) did not display uridine ki-nase activity, suggesting a different role for AtTTM1.Furthermore, Arabidopsis encodes five UK/uracilphosphoribosyl transferase genes (AtUKL1–5), whichcan form uridinemonophosphate from uracil or uridine(Islam et al., 2007). Since we confirmed the uridine ki-nase activity of AtUKL1 in this study, it is likely thatAtTTM1 acts on some other substrate.

Both AtTTM1 and 2 displayed low activity on ATP,ADP, or PPPi substrates, while high affinity was ob-served for PPi with a Km of 17 mM, which is comparableto the Km of AtTTM3 or NeuTTM for PPPi (43 mM and21 mM, respectively; Moeder et al., 2013; Delvaux et al.,2011). We tested several biologically relevant diphos-phates such as thiamine diphosphate (Jordan, 2007),ADP ribose (Adams-Phillips et al., 2010) and NADH(Ishikawa et al., 2010), all of which were not hydrolyzedby AtTTM1 and AtTTM2 (Ung and Yoshioka, unpub-lished data). Thus, it remains to be determined whetherPPi is the biological substrate and whether there areother diphosphate substrates forAtTTM1 andAtTTM2.

Both proteins possess a proper P-loop kinase domainwith Walker A, Walker B, and lid domains (Leipe et al.,2003). The CYTH domain, on the other hand, lacks thesignature EXEXK domain (TYILK). Furthermore, anumber of conserved basic and acidic residues facinginto the b-barrel are not conserved. These features arealso conserved in TTM1/2 orthologs in other plantspecies, indicating that they may be relevant for theircatalytic activity. AtTTM1 and 2 are the first TTMproteins that have been reported to possess activity forPPi, where TTM proteins have only been reported toexclusively catalyze the hydrolysis of triphosphatecompounds (Bettendorff and Wins, 2013). Iyer andAravind (2002) suggested that the altered CYTH do-main in AtTTM1/2 and their ortholog in D. discoideummay have lost its catalytic activity and serves as an al-losteric binding site to modify the activity of the kinase

domain. Alternatively, the CYTH domain may serve asa binding site for a substrate that is phosphorylated bythe P-loop kinase domain.

PPi is the byproduct of a variety of biosyntheticreactions. Removal of PPi prevents these reactionsfrom reaching equilibrium and plays an importantrole in maintaining the direction of these reac-tions (Maeshima, 2000). Arabidopsis encodes sixsoluble inorganic PPases and three membrane-boundH+-translocating PPases (Schulze et al., 2004; Ferjaniet al., 2011). The H+ translocating PPases are locatedin the tonoplast or the Golgi apparatus, while thesoluble PPases are located in the cytosol or chloro-plast (AtPPa6; Schulze et al., 2004). None of thesegenes are up-regulated in senescing leaves likeAtTTM1. Therefore, it is possible that AtTTM1 mightspecifically contribute to maintain low PPi levelsduring senescence. Its function could be to removePPi, which may be necessary to drive a reaction at themitochondrial outer membrane. Further analysis ofATTM1’s molecular mechanism is in progress.

ttm1 and ttm2 knockout lines displayed distinctphenotypes in senescence and disease resistance, re-spectively. Furthermore, phylogenetic analysis indi-cates that most dicotyledonous plants maintain TTM1and TTM2 paralogs (Supplemental Fig. S1) that fall intoseparate clades, further supporting the notion of dis-tinct roles of these genes in plants. However, consid-ering the high homology in their catalytic region (98%and 91% similarity in the uridine kinase and CYTHdomains, respectively), it was of question whether theywould act on the same in vivo substrate. Therefore, wetested whether AtTTM2, under control of the AtTTM1promoter, could complement the ttm1 mutant pheno-type and vice versa. Both paralogs could clearly com-plement the ttm1 mutant phenotype, while bothconstructs under control of the AtTTM2 promoter onlyexhibited a partial rescue. This is probably due to theintricate nature of the Hpa infection assay. Anotherexplanation could be that the cDNA clones used lacksome regulatory element compared to a genomic clone.This is supported by the fact that the pTTM2:TTM2construct also complemented poorly. However, ourdata from the promoter swap experiments and the factthat overexpression of both AtTTM1 and 2 in N. ben-thamiana caused a cell death phenotype in the darkstrongly suggest that the in vivo substrate is likelysimilar or identical for both proteins. At this time, wecannot explain why these two genes can complementeach other yet their mutant phenotypes are distinctlydifferent. Further studies into their biochemical prop-ertiesmay help to answer this question. It is well knownthat autophagy plays an important role in bothpathogen-induced PCD and senescence (Hofius et al.,2017; Yoshimoto et al., 2009). Autophagy can playprosurvival or prodeath roles (Hofius et al., 2017),similar to what we see for AtTTM2 and AtTTM1, re-spectively. Potential roles for AtTTM1 and 2 in au-tophagy may explain the different functions insenescence and pathogen resistance. Further studies

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on a possible role of AtTTM1 and 2 in autophagy areunderway.In summary, we present evidence suggesting that

AtTTM1 and AtTTM2 have distinctive biological rolesand that the spatial and temporal differences in theirexpression determine the different functions duringsenescence and disease resistance, respectively. Withthis study, we completed the basic characterization ofall three Arabidopsis TTMs. These proteins exist in alldomains of life, yet the biological role of most familymembers is not clear, and until recently there was noinformation available about their role in plants. Thus,we have laid the groundwork for the further study ofplant TTM genes. Elucidation of the molecular mecha-nisms underlying the involvement of AtTTM1 andAtTTM2 in these processes is underway.

MATERIALS AND METHODS

Plant Growth Conditions and Pathogen Assays

Arabidopsis (Arabidopsis thaliana) accession Columbia (Col) plants weregrown in Sunshine Mix in a growth chamber at 22°C, 60% relative humidity,and;140mEm22 s21 with a 9-h photoperiod. Seven to ten-day-old Arabidopsisplants were infected with Hyaloperonospora arabidopsidis (Hpa). Spore counts of8 3 105 cells mL21 and 2 3 105 cells mL21 were used for Emco5 and Emwa1isolates, respectively. Seedlings were then infected via drop inoculation and leftin a growth chamber at 16°C, .90% relative humidity for 7 to 10 d beforedisease assessment.

Confirmation of T-DNA Insertion Knockout Lines

The SALK line, SALK_079237 (ttm1-1) and the GABI-Kat line, GABI_672E02(ttm1-2), were obtained from the SALK Institute and Max Planck Institute ofPlant Breeding Research (Alonso et al., 2003; Kleinboelting et al., 2012), re-spectively. Homozygous plants (Arabidopsis accession Columbia) were iso-lated using gene-specific primers for ttm1-1 (229RP, 229LP) and for ttm1-2(980Seq-F, SK73980R1) in combination with the T-DNA specific primers for theSALK line, LBb1-F, and the GABI-Kat line, GABIKAT-TDNA-F. Semiquanti-tative RT-PCR was then performed on cDNA from both ttm1 lines to confirmthe knockout status (Supplemental Fig. S5) using full-length TTM1 primers(980RT-F, 732RT-R). Expression was normalized to the expression of b-tubulin(At-Tub-F, At-Tub-R). Sequencing confirmed T-DNA insertion locations to be atthe 1067-bp position (end of exon 3) and 2693-bp position (middle of exon 9) ofttm1-1 and ttm1-2, respectively (Supplemental Fig. S5). Primer sequences arelisted in Supplemental Table S2.

Trypan Blue Staining

Trypan blue staining of seedlings was performed as previously described(Yoshioka et al., 2001).

Confocal Microscopy

For visualization of themitochondria in Arabidopsis root cells, 4- to 10-d-oldseedlings were soaked in 50 nM MitoTracker Orange (Invitrogen) dissolved insterilized water for 1 min at 23°C. For FM4-64 staining, 4- tp 10-d-old seedlingswere incubated in half-strength MS-1% Suc medium supplemented with 1 mM

FM4-64 (Invitrogen) for 4 h at 23°C. For drug treatments, FM4-64-stained rootswere treated with 50 mM brefeldin A (Sigma) for 30 min or 33 mM Wortmannin(Sigma) for 1 h. After being washed with water, root epidermal cells in thetransition zone were observed using LSM780 (Carl Zeiss). Emissions from YFP(490–553 nm), MitoTracker Orange (562–758 nm), and FM4-64 (562–695 nm)excited by 488-nm or 561-nm laser were detected with the Plan-Apochromat633/1.40 oil immersion objective lens.

For confocal microscopy of Nicotiana benthamiana leaves, 1-cm sectionsof Agrobacterium-infiltrated leaves were excised 24 h postinfiltration for

microscopy using the Leica TCS SP5 confocal system (LeicaMicrosystems). YFP(520–590 nm) or chloroplast autofluorescence (650–700 nm) was detected underthe 403 oil immersion objective lens (numerical aperture 1.40) with 23 zoomusing the 514-nm OPSL laser set to 33%.

RNA Extraction and RT-PCR

RNAextractionwas carried out using the TRIzol reagent (Life Technologies),according to the manufacturer’s instructions. Reverse transcriptase (RT)-PCRwas performed using cDNA generated by SuperScript II Reverse Transcriptase(Life Technologies) according to the manufacturer’s instructions. Expression ofPR1, CAB6, SAG12, and SAG13 was visualized by gel electrophoresis of sam-ples after RT-PCR with the following RT primers: AtPR1-F, AtPR1-R, AtCAB6-F, AtCAB6-R, AtSAG12-F, AtSAG12-R, AtSAG13-F, and AtSAG13-R. Allprimer sequences are listed in Supplemental Table S2.

Quantitative Real-Time PCR

Quantitative real-time PCR was performed using Fast SYBR Green MasterMix (Life Technologies). The expression ofArabidopsis geneswas normalized tothe expression of AtEF1A (elongation factor1-alpha). All primer sequences arelisted in Supplemental Table S2.

BTH Treatments

Seven- to ten-day-old Arabidopsis seedlings were treated with 200 mM BTH.RNA was then isolated from pooled leaf tissue samples 48 h after treatment.

Dark Senescence Assay

A combination of nonsenescent leaves 3, 4, 5, and 6 of 4- to 5-week-old plantswere detached and floated on tap water in petri dishes in the dark for thespecified amount of time. Leaf samples were then weighed, frozen in liquid N2,and crushed in 80% acetone (v/v), 25 mM HEPES, pH 7.5. Total chlorophyllcontent was quantified by measuring the absorbance of chlorophylls A and Busing a spectrophotometer and the equation, total chlorophyll content = 17.76(A646) + 7.34 (A663) (Porra et al., 1989), followed by normalization to freshweight.

Plant Complementation Analysis

Full-length AtTTM1 genomic sequence was cloned from the promoter re-gion (905-bp upstream of the ATG start codon) to the end of the 39 UTR regionusing the primers, TTM1-genomic-F, and TTM1-genomic-R, into pORE-O1(Coutu et al., 2007). For the confocal analysis ttm1 plants were transformedwith a proTTM1:YFP-TTM1 construct in the pORE R2 plasmid (Coutu et al.,2007). Arabidopsis Columbia wild-type plants were stably transformed byAgrobacterium tumefaciens-mediated transformation using the floral dip method(Clough and Bent, 1998). Primer sequences are listed in Supplemental Table S2.

Promoter Swap Analysis

For the promoter swap analysis, full-lengthAtTTM1 orAtTTM2 cDNAwitha C-terminal HA tag was cloned using TTM1swap-F and TTM1swap-R andTTM2swap-F and TTM2swap-R, respectively. The inserts were used to replacethe uidA gene in the pORE R2 plant expression vector carrying the AtTTM1 orAtTTM2 promoter. ttm1-1 and ttm2-1 plants were stably transformed by A.tumefaciens-mediated transformation using the floral-dip method for each clone(Clough and Bent, 1998). This resulted in four transgenic lines: pTTM1:TTM1(ttm1-1), pTTM1:TTM2 (ttm1-1), pTTM2:TTM1 (ttm2-1), and pTTM2:TTM2(ttm2-1). Primer sequences are listed in Supplemental Table S2.

Agrobacterium-Mediated Transient Expression

N. benthamiana was grown on Sunshine mix soil (Sun Gro HorticultureCanada) in a growth chamber under a 9/15-h light/dark regimen at 22°C (day)and 20°C (night; 60% relative humidity, and approximately 140 mE m22 s21).Transient expression was performed via infiltration of N. benthamiana with A.tumefaciens (strain GV2260) as described previously (Urquhart et al., 2007).Agrobacterium carrying CaMV35S:HC-Pro from Tobacco etch virus (labeled TEV

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in the figures) was infiltrated alone or coinfiltrated with constructs as describedin the figure captions. The YFP-TTM constructs were in pEARLEYGATE 104 -(Earley et al., 2006)

Protein Expression in Escherichia coli

Coding regions of AtTTM1 and AtTTM2 were cloned into the pGEX-6P-1vector from Arabidopsis Columbia ecotype cDNA using the primers pGEX-TTM1-F, pGEX-TTM1-R, pGEX-TTM2-F, and pGEX-TTM2-R, which excludethe annotated C-terminal transmembrane domain and end at S621 and D648 ofAtTTM1 and AtTTM2, respectively. Each plasmid was introduced into theE. coli BL21 codon plus cells and grown overnight in Luria-Bertani medium at37°C. The overnight culture was used to seed a larger volume of Autoinductionmedium containing 13 NPS solution (25 mM (NH4)2SO4, 50 mM KH2PO4, and50 mM Na2HPO4) and 13 5052 solution (0.05% Glc, 0.2% a-lactose, and 0.5%glycerol), which was grown at 37°C for 3 to 4 h until OD600 = 0.4. The tem-perature was then lowered to 18°C overnight before harvesting the cells bycentrifugation at 4°C.

Protein Extraction

E. coli cultures were centrifuged and pellets were resuspended in13 phosphate-buffered saline, pH 7.5 (137 mM NaCl, 2.7 mM KCl, 10 mM

Na2HPO4, and 1.8 mM KH2PO4), containing 1 mM phenylmethylsulfonyl fluo-ride, 1 mM dithiothreitol, and 10 mg mL21 DNase I. Cell suspensions were in-cubated on ice for 30 min before cell lysis by French press at 1000 psi. Solublefractions were obtained by centrifugation and subjected to column purificationusing DE52 cellulose (Sigma) and GSH agarose (Sigma). Purified proteinsamples were eluted using 10 mM reduced glutathione, quantified usingBradford reagent, and stored at 280°C until use.

Malachite Green Assay

Detection of free phosphates was performed as previously described (Bernalet al., 2005; Moeder et al., 2013).

Statistical Analysis

A two-tailed Student’s t test was performed for all comparisons between twosample groups. A P-value of less than 0.05 was used to denote significance.Fisher’s exact test was performed for all comparisons between two sampleswith multiple groups.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accession num-bers: Arabidopsis thaliana (At)—AtTTM1 (At1g73980), AtTTM2 (At1g26190),AtEF1A (At5g60390), b-tub (At5g23860), AtCAB6 (At3g54890), AtSAG12(At5g45890), AtSAG13 (At2g29350); Brassica napus (Bn)—BnTTM1 (Bra008117),BnTTM2a (Bra011014), BnTTM2b (Bra012464); Glycine max (Gm)—GmTTM1a(Glyma05g07610), GmTTM1b (Glyma17g09080), GmTTM2a (Gm1g09660),GmTTM2b (Gm2g14110); Cucumis sativus (Csa)—CsaTTM1 (Cucsa.198420),CsaTTM2 (Cucsa.284210);Citrus sinensis (Csi)—CsiTTM1 (Csi:orange1.1g006094m),CsiTTM2 (Csi:orange1.1g038045m); Theobroma cacao (Tc)—TcTTM1 (Thecc1EG014447t1),TcTTM2 (Thecc1EG011378t1).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Domain structure and evolutionary relationshipof TTM family members.

Supplemental Figure S2. Amino acid sequence alignment of AtTTM1 andAtTTM2.

Supplemental Figure S3. AtTTM1 and AtTTM2 do not exhibit uridinekinase activity.

Supplemental Figure S4. Expression patterns of AtTTM1 and AtTTM2.

Supplemental Figure S5. T-DNA insertion line analysis.

Supplemental Figure S6. Whole-plant dark-induced senescence.

Supplemental Figure S7. YFP-TTM1 complementation lines.

Supplemental Figure S8. YFP-TTM1 is not localized to the Golgi appara-tus or endosome.

Supplemental Figure S9. Subcellular localization of YFP-AtTTM1 andYFP-AtTTM2.

Supplemental Figure S10. Pathogen infection phenotype of promoterswap constructs.

Supplemental Table S1. Genes that are coexpressed with AtTTM1.

Supplemental Table S2. Primers used in this study.

Received May 26, 2017; accepted July 15, 2017; published July 21, 2017.

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AtTTM1 Is Involved in Dark-Induced Leaf Senescence

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