Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This 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 Moeder a,3 a

Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3B2, Canada Center for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, ON M5S 3B2, Canada c Division of Cellular Dynamics, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan d Department of Basic Biology, Graduate University for Advanced Studies, Okazaki, Aichi 444-8585, Japan b

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

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises two groups of enzymes, RNA triphosphatases and CYTH phosphatases (CyaB adenylate cyclase, thiamine triphosphatase) that possess 1 This article was supported by a Discovery Grant from the Natural Science and Engineering Research Council of Canada (grant no. PGPIN-2014-04114), Canadian Foundation for Innovation, and Ontario Research Fund to K.Y., grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24114003 and 15H04382) to T.U., and a graduate student fellowship from the Ontario government to H.U. 2 These authors contributed equally to the article. 3 Address correspondence to [email protected] and [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Keiko Yoshioka ([email protected]). W.M, H.U., and K.Y. designed the research; H.U., P.K., K.E., and T.U. performed the research; H.U., P.K., W.M., and K.Y. analyzed the data; W.M., H.U., and K.Y. wrote the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.17.00700

common characteristics in their catalytic sites (Iyer and Aravind, 2002; Gong et al., 2006). Members of this superfamily are able to hydrolyze a variety of triphosphate substrates, giving them important roles in cAMP formation, mRNA capping, and secondary metabolism (Iyer and Aravind, 2002; Gallagher et al., 2006; Gong et al., 2006; Song et al., 2008). Most TTMs possess a unique tunnel structure composed of eight antiparallel beta strands forming a beta barrel and a characteristic EXEXK motif (where X is any amino acid), which is important for catalytic activity (Lima et al., 1999; Iyer and Aravind, 2002; Gallagher et al., 2006). In addition, TTMs also share the requirement of a divalent metal cation cofactor, usually Mg2+ or Mn2+ (Bettendorff and Wins, 2013). While the catalytic activity of some TTMs has been elucidated, the biological function of most TTM proteins is unknown. However, it appears they have acquired the ability to act on a diverse range of nucleotide and organophosphate substrates (Iyer and Aravind, 2002; Bettendorff and Wins, 2013). Known functions of TTMs include fungal and protozoan RNA triphosphatases (Cet1; Lima et al., 1999; Gong et al., 2006), bacterial

Plant PhysiologyÒ, September 2017, Vol. 175, pp. 473–485, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved.

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adenylate cyclases (CyaB from Aeromonas hydrophila and YpAC-IV from Yersinia pestis; Sismeiro et al., 1998; Gallagher et al., 2006), and mammalian thiamine triphosphatases (Lakaye et al., 2004; Song et al., 2008). More recently, tripolyphosphatase activity was discovered for CthTTM from Clostridium thermocellum and NeuTTM from Nitrosomonas europaea, highlighting the functional diversity of this superfamily (Keppetipola et 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 a singular CYTH domain and one with a CYTH domain fused to a P-loop kinase domain (Iyer and Aravind, 2002). In Arabidopsis (Arabidopsis thaliana), there are three TTM family members, AtTTM1, 2, and 3: AtTTM1 and 2 belong to the latter type, possessing a uridine kinase (UK) domain in addition to the CYTH domain, while AtTTM3 belongs to the former type, possessing only a singular CYTH domain (Supplemental Fig. S1). Previously, we demonstrated that AtTTM3 exhibits tripolyphosphatase activity and may play a role in root development (Moeder et al., 2013), whereas AtTTM2 displays pyrophosphatase (PPase) activity and is a negative regulator of pathogen defense responses (Ung et al., 2014). Interestingly, publicly available microarray data show significantly different expression patterns for AtTTM1 and AtTTM2. This suggests that while AtTTM1 and AtTTM2 possess the same domain arrangement, these genes may play distinct roles where AtTTM1 may be involved in leaf senescence. Leaf senescence is an active and highly regulated process where nutrients are remobilized to other growing tissues of the plant. Individual cells within a leaf undergo metabolic changes in order to dismantle each component before programmed cell death (PCD) occurs and sink-source relationships begin to transition. The initiation of leaf senescence naturally occurs by aging but can also be induced by a range of external factors such as drought, darkness, and hormones (abscisic acid, ethylene), resulting in the visible loss of chlorophyll or yellowing, since the chloroplasts are the first organelles to be disassembled (Weaver et al., 1998; Breeze et al., 2011). Leaf senescence is generally believed to be a special form of PCD, which shares some, but not all, of the characteristics of PCD (van Doorn and Woltering, 2004). Usually a carefully orchestrated dismantling of the chloroplast is one of the first events in the senescence process (Keech et al., 2007). Other organelles, such as mitochondria and peroxisomes, remain active much longer and provide energy for the cell as well as take over a number of metabolic tasks during this process (Keech et al., 2007; Chrobok et al., 2016). While yeast and animals possess mitochondrial PPases (Lundin et al., 1991; Curbo et al., 2006), so far no mitochondrial PPase has been identified in plants. But generally PPases remove pyrophosphate (PPi) that is created as a byproduct of anabolic processes such as nucleic acid, protein, and carbohydrate synthesis (Gómez-García 474

et al., 2006). Removal of PPi is necessary to prevent the inhibition of thermodynamically unfavorable reactions (Maeshima 2000). Another function could be to keep cytosolic [PPi] levels low, since high [PPi] is toxic (Cooperman et al., 1992). Here, we show that AtTTM1 is localized to the mitochondrial outer membrane and, just like its close paralog AtTTM2, displays in vitro PPase activity. AtTTM1 is a positive regulator of dark-induced and natural leaf senescence. It shares high sequence similarity with its paralog, AtTTM2, and AtTTM2 can functionally complement the knockout phenotype of AtTTM1. Moreover, AtTTM2 overexpression can mimic AtTTM1’s cell death-inducing function. In spite of their similarities, these two genes do not appear to be involved in the same biological processes. Rather, it is their different gene expression patterns that dictate their distinct biological roles.

RESULTS AtTTM1 and AtTTM2 Share Many Common Properties

Arabidopsis possesses three genes that are annotated as CYTH domain/TTM proteins. AtTTM3 comprises only a CYTH domain, while the CYTH domain follows an N-terminal uridine kinase domain in AtTTM1 and AtTTM2. AtTTM1 and 2 also possess a coiled-coil domain and a transmembrane domain at the C-terminal end (Supplemental Fig. S1A). AtTTM1 and 2 share high amino acid sequence similarity with over 92% sequence similarity and approximately 66% sequence identity, which is even higher in the UK (81% identity) and CYTH domains (73% identity; Supplemental Fig. S1B). This suggests that AtTTM1 and 2 have either identical or very similar enzymatic properties. Most other dicot plant species also encode TTM1 and TTM2 orthologs, which fall into two distinct clades, indicating conserved distinct functions for AtTTM1 and 2 (Supplemental Fig. S1C). Enzymatic Properties of AtTTM1 and AtTTM2

Plant CYTH domain proteins have been annotated as adenylate cyclases based on their sequence similarity to the adenylate cyclase from Aeromonas hydrophila (Sismeiro et al., 1998; Iyer and Aravind, 2002). However, all three recombinantly expressed TTM proteins from Arabidopsis did not display adenylate cyclase activity (Moeder et al., 2013; Ung et al., 2014). Rather, we previously demonstrated that AtTTM3 displays strong tripolyphosphatase activity with a weaker affinity for nucleotide triphosphates (Moeder et al., 2013). On the other hand, AtTTM2 showed a strong preference for pyrophosphate (PPi; Ung et al., 2014). AtTTM1 revealed the same properties as AtTTM2, with highest activity for PPi and weaker activities for ATP, ADP, and tripolyphosphate (PPPi; Fig. 1A). Further biochemical analyses revealed a slight preference for alkaline pH Plant Physiol. Vol. 175, 2017

AtTTM1 Is Involved in Dark-Induced Leaf Senescence

min21 mg21) for AtTTM1 and AtTTM2, respectively (Fig. 1, D and E). The N-terminal kinase domain is a typical P-loop kinase, which was annotated as a uridine/cytidine kinase. It has conserved Walker A, Walker B, and lid module motifs (Supplemental Fig. S2; Leipe et al., 2003). Thus, the uridine kinase activity of recombinant proteins was tested. Both AtTTM1 and 2 did not display activity on a uridine substrate, while a known Arabidopsis uridine kinase (AtUKL1; Islam et al., 2007) produced uridine monophosphate as expected (Supplemental Fig. S3). This suggests that the kinase domain acts on a different substrate than uridine. AtTTM1 and AtTTM2 Are Differentially Expressed across Various Tissues

Since AtTTM1 and AtTTM2 both displayed similar catalytic activities, it raised the question whether they are redundant or whether they have taken on different biological roles. Thus, the expression patterns of AtTTM1 and AtTTM2 were examined using the Bio-Analytic Resource (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi; Schmid et al., 2005; Winter et al., 2007). While AtTTM2 was predominantly expressed in the shoot apices of inflorescences, AtTTM1 appeared to be ubiquitously expressed in all tissues, with strong expression detected in senescent leaves (Supplemental Fig. S4). This suggests that while the same catalytic activity was detected for both AtTTM1 and AtTTM2, the biological roles they play may be different. ttm1 Plants Do Not Show Altered Disease Resistance Figure 1. Both AtTTM1 and AtTTM2 display pyrophosphatase activity. A, Substrate specificity of AtTTM1 and AtTTM2. Enzymatic activity was tested with various phosphate compounds. Reactions with PPi, ATP, and PPPi 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 were performed in the same conditions except with 0.03 mM substrate to reduce background phosphate readings. Glutathione S-transferase (GST) served as a negative control. Each bar represents the mean 6 SE (n = 3). The experiment was repeated more than three times with similar results. B, Pyrophosphatase activity as a function of pH in the presence of 0.5 mM PPi, 2.5 mM Mg2+, and 2 mg of protein. Each data point represents the mean 6 SE (n = 3). The experiment was repeated twice with similar results. C, Divalent cation cofactor specificity of pyrophosphatase activity. Reactions were performed at pH 9 in the presence of 0.5 mM PPi, 2.5 mM cation cofactor, and 2 mg of protein. Each bar represents the mean 6 SE (n = 3). The experiment was repeated twice with similar results. (D and E) Pyrophosphatase activity as a function of pyrophosphate concentration. Reactions were performed at pH 9 in the presence of 0 to 600 mM PPi, 2.5 mM Mg2+, and 2 mg of AtTTM1 (D) and AtTTM2 (E) protein. Each data point represents the mean 6 SE (n = 3). The experiment was repeated twice with similar results.

(pH 8–9) as well as a strong cofactor preference for Mg2+ (Fig. 1, B and C). The Km for pyrophosphate was determined to be 16.7 6 5.2 mM (Vmax = 284 6 19 nmol min21 mg21) and 17 6 3.2 mM (Vmax = 366 6 15 nmol Plant Physiol. Vol. 175, 2017

The differences in the expression patterns of AtTTM1 and 2 prompted us to further analyze the differences in their transcriptional regulation. It has been shown that AtTTM2 is a negative regulator of the SA-dependent amplification loop for pathogen defense responses and is also transcriptionally suppressed upon pathogen infection and treatments with the pathogen-associated molecular pattern, flg22 (flagellin peptide), the defense hormone salicylic acid (SA) or the biological analog of SA, benzothiadiazole (BTH; Ung et al., 2014). Therefore, we monitored the transcript levels of AtTTM1 after infection with the avirulent Hyaloperonospora arabidopsidis (Hpa) isolate, Emwa1. In contrast to AtTTM2, AtTTM1 expression was not down-regulated upon pathogen infection while the Pathogenesis-related1 (PR1) gene, a marker of pathogen resistance activation, was strongly induced, as expected (Fig. 2A; Ung et al., 2014), indicating a fundamental difference in the transcriptional regulation between AtTTM1 and 2. This result suggests that AtTTM1 does not play the same role as AtTTM2 in pathogen defense. Next, two allelic T-DNA insertion knockout lines of AtTTM1, ttm1-1 and ttm1-2, were isolated for phenotypic characterization. Both mutants exhibited no discernible morphological difference to Columbia wild 475

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type (Col; Supplemental Fig. S5). Following infection with the avirulent Hpa isolate, Emwa1, as reported prior, ttm2 mutants developed significantly more hypersensitive response (HR) cell death compared to wild type, indicating enhanced defense activation (Fig. 2, B and C; Ung et al., 2014). On the other hand ttm1 mutants displayed a subtle but statistically significant attenuation of HR cell death, but no effect on fungal growth was observed. Furthermore, infection with the virulent Hpa isolate, Emco5, revealed that ttm2 plants displayed significantly less growth of the pathogen, while ttm1 mutants showed no significant difference from wildtype plants (Fig. 2, D and E). These data suggest that AtTTM1 does not play the same role as AtTTM2 in pathogen defense. ttm1 Displays Delayed Senescence Phenotypes

Since transcriptional regulation of AtTTM2 is tightly connected to its biological role (Ung et al., 2014), we investigated the transcriptional regulation of AtTTM1 in order to gain insight into its biological role. A noticeable increase in AtTTM1 transcript levels was observed in senescing leaves (Supplemental Fig. S4). Furthermore, several coexpressed genes were senescence-associated genes, such as a caspase-like protease, gamma vacuolar processing enzyme (g-VPE), receptor-like protein kinase1, SENESCENCE-ASSOCIATED GENE SAG12, and a Cys proteinase (Supplemental Table S1; Expression Angler, http://bar.utoronto.ca/ntools/cgi-bin/ ntools_expression_angler.cgi; Toufighi et al., 2005). Therefore, we hypothesized that AtTTM1 is involved in leaf senescence and assessed transcriptional changes during senescence using the well-established darkinduced senescence assay (Riefler et al., 2006). We first validated the microarray data by monitoring the expression levels of AtTTM1 in detached leaves over the course of 7 d in darkness. As shown in Figure 3, AtTTM1 transcript levels were already over 3-fold increased after 1 d and continued to increase until 7 d in darkness,

Figure 2. ttm1 does not show enhanced immunity to Hyaloperonospora arabidopsidis. A, AtTTM1 expression is not suppressed by pathogen infection. Quantitative real-time PCR analysis of AtTTM1 (left) or PR1 (right, as a control of infection) expression in Hyaloperonospora arabidopsidis (Hpa) isolate, Emwa1-infected (Emwa1), or water-treated (H2O) cotyledons 7 d after infection with Hpa. Transcripts were normalized to AtEF1a. Shown are the averages of three independent experiments. Each bar represents the mean 6 SE (n = 3). An asterisk indicates significant difference to the H2O control, (Student’s t test, p , 0.001). All samples represent a pool of 14 to 20 seedlings. B Infection phenotype of Columbia wild type (Col), ttm1-1, and ttm2-1 mutant plants 10 d after infection with the avirulent Hpa isolate, Emwa1. Shown is Trypan blue staining of infected cotyledons (Cot) revealing HR 476

cell death (white arrows) and some hyphae (red arrows in Col and ttm1) and uninfected first true leaves (TL) revealing enhanced HR in ttm2 plants. Scale bar = 250 mm. C, HR index of Hpa isolate Emwa1 infection. Stained leaves were microscopically examined and assigned to different classes (see right; n = 45). The experiment was repeated three times with similar results. tm2-1 displayed significantly more HR than Col (Fisher’s exact test, P = 0), while ttm1-1 showed less HR than Col wild type (P = 0.005). D, Infection phenotype of Col wild type, ttm1-1, and ttm2-1 mutant plants 12 d after infection with virulent Hpa, isolate Emco5. Shown is Trypan blue staining of infected cotyledons revealing hyphae (white arrows) and oospores (red arrows) in wild type and ttm1-1 and reduced hyphal growth in ttm2-1. Scale bar = 250 mm. E, Disease index of Hpa isolate Emco5 infection. Stained leaves were microscopically examined and assigned to different classes (right; n = 30). A significant difference was detected between ttm2-1 and Col (Fisher’s exact test, P , 0.0001), but not between ttm1-1 and Col. Highly significant differences are marked by an asterisk. The experiment was repeated four times with similar results. Plant Physiol. Vol. 175, 2017

AtTTM1 Is Involved in Dark-Induced Leaf Senescence

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

whereas AtTTM1 levels rose to over 8-fold. On the other hand, AtTTM2 transcript levels showed only a mild increase after 1 d but did not increase further over the course of the experiment (Fig. 3). Next, we monitored the dark-induced senescence phenotype in ttm1 mutants. Leaves 3 to 6 of 4- to 5-week-old plants were detached and floated on water in the dark. Strikingly, they consistently displayed less chlorophyll loss during the course of the experiment (Fig. 4A). This difference was observed as early as 5 d after dark treatment and was most dramatic 7 d after. Measurement of the total chlorophyll content confirmed this observation quantitatively (Fig. 4B). To confirm that the difference in chlorophyll retention was indeed due to the absence of AtTTM1, a genomic fragment comprising the promoter region and the AtTTM1 gene was introduced into the ttm1 knockout mutant background, and two independent transgenic lines were analyzed. Complementation of the ttm1 mutant plants with wild-type AtTTM1 rescued the delayed senescence phenotype of ttm1, returning chlorophyll retention 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 several senescence markers were monitored. CAB6 and SAG13 are known to be down-regulated and up-regulated, respectively, during the transition from vegetative growth to senescence, whereas SAG12 is senescence specific and is strongly induced when this process is activated (Lohman et al., 1994). In ttm1 mutant leaves, the transcriptional down-regulation of CAB6 was delayed by 2 d compared to wild type over the course of 7 d in darkness (Fig. 4E). In addition, SAG13 transcript levels were visibly lower in ttm1 mutant leaves compared to wild type. Furthermore, SAG12 expression was starkly induced at 5 d after darkness in wild-type leaves but did not express in ttm1 mutant leaves until 7 d (Fig. 4E). Taken together, these data suggest that AtTTM1 is a positive regulator of dark-induced senescence. Plant Physiol. Vol. 175, 2017

To investigate whether ttm1 mutants also display delayed senescence in intact plants, we first placed whole plants in the dark and assess the chlorophyll content after 5 d. Similar to our detached leaf assay, we also observed less chlorophyll loss in intact ttm1 mutant plants (Supplemental Fig. S6). To further test whether AtTTM1 also plays a role in natural senescence, plants were grown under 16 h light and monitored for first signs of leaf yellowing. At 5 weeks, wild-type plants started to display first signs of yellowing on the oldest leaves, while comparable leaves of ttm1 plants remained green (Fig. 5A). The ttm1/TTM1 complementation plants showed the same timing of yellowing as wild-type plants. The chlorophyll content of these leaves was also significantly higher in ttm1 plants compared to the wild type and ttm1/TTM1 complementation plants (Fig. 5B). ttm2 Does Not Show a Senescence Phenotype, and the ttm1 ttm2 Double Mutant Does Not Exhibit Additive Effects

Transcriptional analysis of AtTTM2 upon darkinduced senescence treatment suggested that AtTTM2 is not involved in senescence and that its biological role is different from that of AtTTM1. To further validate this point, we first analyzed the dark-induced senescence phenotype of ttm2 mutants. As shown in Figure 6, ttm2 plants showed the same degree of chlorophyll loss as wild-type leaves. Combined with the absence of a disease-resistance phenotype in ttm1 plants, this indicates that AtTTM1 and 2 are not biologically redundant. To further address this point and also investigate possible synergistic effects, double-knockout lines were generated by cross pollination, and their phenotypes were compared to their respective single-knockout lines. The ttm1 ttm2 double mutant did not display any morphological differences compared to wild type (Fig. 6A). After 7 d in the dark, the ttm1 ttm2 double mutant displayed the same chlorophyll retention as the ttm1 single mutant, while the ttm2 single mutant behaved like wild type, suggesting that only AtTTM1, but not AtTTM2, 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 ttm1 ttm2 double mutant. The double mutant displayed a phenotype similar to the ttm2 single mutant, while ttm1 plants behaved like Col wild type plants (Fig. 6, D and E). These data suggest that AtTTM1 is involved in senescence, whereas AtTTM2 plays a role in pathogen defense, further indicating their involvement in independent biological processes. AtTTM1 Is Localized to the Mitochondrial Outer Membrane

To determine the subcellular localization of AtTTM1, ttm1 plants were transformed with a pTTM1:YFP-TTM1 construct. To confirm that the fusion protein is properly localized and functional, several independent lines were assessed in the dark-induced senescence 477

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complementation assay (Supplemental Fig. S7A). Three independent lines displayed the same complementation phenotype as the complementation line without YFP (Fig. 4D). Four- to ten-day-old seedlings were then used for confocal microscopy. YFP-AtTTM1 displayed a punctate pattern in two independent transgenic lines (Fig. 7; Supplemental Fig. S7B). In order to determine which subcellular compartment these puncta represent, we first analyzed the effect of wortmannin and brefeldin A. The YFP pattern was not affected by these inhibitors, indicating that AtTTM1 is not localized to the Golgi, trans-Golgi network, or endosome (Supplemental Fig. S8). On the other hand, the YFP pattern clearly colocalized with the MitoTracker dye (Fig. 7; Supplemental Fig. S7), suggesting a mitochondrial localization. Furthermore, higher magnification images showed that the YFP signal surrounds the MitoTracker signal, indicating a localization in the mitochondrial outer membrane (Fig. 7B). Indeed, AtTTM1 has a transmembrane domain at its C terminus (Fig. 7C) and has been predicted to be a tailanchored protein that is localized to mitochondria (Kriechbaumer et al., 2009). A pTTM1:YFP-TTM1 DTM construct completely lost the mitochondrial localization and instead was found in the cytosol (Supplemental Fig. S7B). This construct also failed to complement the ttm1 senescence phenotype (Supplemental Fig. S7A), suggesting that the mitochondrial localization is required for its biological function. On the other hand, a YFP signal from plants transformed with the pTTM2:YFP-TTM2 construct could not be detected, most likely due to the low expression levels of AtTTM2 (Supplemental Fig. S4). Therefore, YFP-tagged AtTTM1 and 2 under the CaMV35S promoter were transiently expressed in N. benthamiana. In both cases a similar punctate pattern was observed for both AtTTM1 and 2 (Supplemental Fig. S9). This and the fact that Kriechbaumer et al. (2009) and Marty et al. (2014) also identified AtTTM2 as a tail-anchor protein suggests that AtTTM2 is also localized to the mitochondrial outer membrane. AtTTM1 and AtTTM2 Can Functionally Complement Each Other

AtTTM1 and AtTTM2 exhibit distinct expression patterns and knockout phenotypes. Furthermore, the double mutant did not display enhanced phenotypes for 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 type and ttm1 mutants were detached and floated for 7 d on water in the dark. Pictures were taken at day 0 and day 7. Scale bar = 1cm. B, Total chlorophyll content was measured 0 and 7 d after dark treatment. Each bar represents the mean 6 SE (n = 3). An asterisk denotes significance 478

difference to Col wild type (P , 0.01). The experiment was repeated three times with similar results. C, Leaves of 5-week-old Col wild type, ttm1-1, and two independent ttm1/TTM1 complementation lines were detached and floated for 7 d on water in the dark. Pictures were taken at day 0 and day 7. Scale bar = 1cm. D, Total chlorophyll content was measured 0 and 7 d after dark treatment. Each bar represents the mean 6 SE (n = 3). Bars marked with different letters indicate a significant difference to Col wild type (P , 0.0001). The experiment was repeated twice with similar results. E, Expression of senescence markers CAB6, SAG12, and SAG13 in detached leaves of Col wild type and ttm1-1 mutant plants after dark treatment. b-tubulin (TUB) served as loading control. Plant Physiol. Vol. 175, 2017

AtTTM1 Is Involved in Dark-Induced Leaf Senescence

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

(Fig. 6A). However, the two genes have very high sequence similarity, all known catalytic amino acid residues are conserved between them (Supplemental Fig. S2), and their subcellular localization is most likely the same. Thus, we asked whether the biological function of the two genes is solely conferred by their expression patterns. To address this question, functional complementation was conducted using four promoter swap constructs: (1) the AtTTM1 promoter followed by either the coding sequence (CDS) of AtTTM1 or AtTTM2 and (2) the AtTTM2 promoter followed by the CDS of either AtTTM1 or AtTTM2. Three independent transgenic lines each were analyzed. The AtTTM1 promoter lines were subjected to the dark-induced senescence assay, revealing that both the AtTTM1 as well as the AtTTM2 CDS can complement the chlorophyll retention Plant Physiol. Vol. 175, 2017

Figure 6. The ttm1 ttm2 double mutant does not exhibit additive effects on senescence and pathogen resistance phenotypes. A, Morphological phenotype of Arabidopsis accession Columbia (Col) wild type, ttm1-1, ttm1-2, and ttm1-1 ttm2-1 plants. Photos show approximately 5-week-old plants. 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 in the 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 different letters indicate a significant difference (P , 0.01). The experiment was repeated three times with similar results. D, Infection phenotype of Col wild type, ttm1-1, ttm1-2, and ttm1-1 ttm2-1 plants 11 d after infection with the virulent Hpa isolate, Emco5. Shown is Trypan blue staining of infected cotyledons revealing hyphae (white arrows) and oospores (red arrows). Scale bar = 250 mm. E, Stained leaves were microscopically examined and assigned to different classes (see below; n = 105–112, from four independent experiments). Fisher’s exact test was performed. Significant differences are marked by an asterisk. 479

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analysis, overexpression of AtTTM2 also induced cell death in the dark (Fig. 8, C and D). Taken together, these data indicate that the in planta enzymatic function of AtTTM1 and AtTTM2 is identical or at least very similar. This suggests that the different expression pattern of these two genes is critical for their specific biological functions.

DISCUSSION

Figure 7. YFP-TTM1 localizes to the mitochondria. A, Ten-day-old seedlings of pTTM1:YFP-TTM1 plants were analyzed by confocal microscopy. The YFP signal localized around the mitochondria stained with 50 nM MitoTracker Orange. Scale bar = 10 mm. B, Higher magnification observation of the mitochondria stained with MitoTracker Orange, which are surrounded by the YFP signal, indicating a localization of AtTTM1 in the mitochondrial outer membrane. Scale bar = 10 mm. C, Domain structure of AtTTM1. (UK = uridine kinase, CYTH = CyaB thiamine triphosphatase domain, CC = coiled-coil domain, TM = transmembrane domain). Bottom, Sequence of the transmembrane domain of AtTTM1 and AtTTM2 (black) and the C-terminal tail (red).

phenotype of ttm1 (Fig. 8A). To confirm that the promoter constructs behaved as expected, quantitative real-time PCR was used to analyze the up-regulation of both AtTTM1 and AtTTM2 under the control of the AtTTM1 promoter after 7 d of dark treatment (Fig. 8B). In the reverse experiment, the AtTTM2 promoter lines were subjected to Hpa infection. Quantitative real-time PCR analysis confirmed that both AtTTM1 and AtTTM2 under the control of the AtTTM2 promoter were similarly down-regulated after treatment with BTH, as previously shown (Supplemental Fig. S10A; Ung et al., 2014). As shown in Supplemental Figure S10B, only one of three pTTM2:TTM2 and two out of three pTTM2:TTM1 independent lines showed a clear rescue of the enhanced disease resistance phenotype of ttm2 (Supplemental Figure S10B). These data indicate the possibility of functional similarity of these proteins. However, since complementation of ttm2 was not seen in all transgenic lines, we further tested transient overexpression of both genes under the control of the CaMV35S promoter in N. benthamiana. We found that transient overexpression of AtTTM1 induced senescence-like cell death 7 d after Agrobacterium infiltration when the plants were kept in the dark (Fig. 8C). However, this cell death was not observed when plants were kept in the light (Fig. 8D), which matches AtTTM1’s function during darkinduced senescence. As suggested by the complementation 480

The TTM superfamily is characterized by an active site situated within a tunnel comprised of antiparallel b-sheets. Furthermore, members of this superfamily act on triphosphate substrates with a strict dependency on a metal cation cofactor. TTM proteins are present in all living organisms, where they have taken up a range of different functions. What they have in common is that their substrates contain triphosphate moieties (Bettendorff and Wins, 2013). Plants are unique in two ways: First, they possess usually three TTM genes, while most other organisms only encode one type (Iyer and Aravind, 2002; Bettendorff and Wins, 2013). Second, AtTTM3 and its orthologs are comprised of only a CYTH domain while AtTTM1 and 2 and their orthologs display an additional N-terminal uridine kinase domain. This fusion of a uridine kinase and a CYTH domain is only seen in plants and members of the slime mold family (Mycetozoa), such as Dictyostelium discoideum (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 two glutamates of the EXEXK motif are not conserved (TYILK in AtTTM1 and 2, IYILK and VYVCK in udkC and D, respectively), as are a number of the conserved basic and acidic residues facing into the b-barrel. Interestingly, the K of the EXEXK domain still is conserved, and all four proteins have a P residue before the modified EXEXK domain (other TTM proteins typically have an aliphatic residue in that position—I, L, or V). We have previously characterized AtTTM3 as a tripolyphosphatase with a potential function in root development (Moeder et al., 2013) and AtTTM2 as a negative regulator of the SA amplification loop during pathogen resistance responses (Ung et al., 2014). Knockout lines of AtTTM1, which shows 65% identity and 92% similarity to AtTTM2 at the amino acid level, did not display enhanced disease resistance, but rather showed a delayed senescence phenotype, as was expected based on our analysis of coexpressed genes. When senescence was induced by dark treatment of detached leaves, ttm1 plants retained chlorophyll longer than wild-type plants. Furthermore, they displayed delayed induction of the senescence marker genes, SAG12 and SAG13, suggesting that AtTTM1 plays a role during the senescence process. The fact that the ttm1 delayed senescence phenotype was also observed after whole-plant dark treatment and during natural senescence clearly indicates that Plant Physiol. Vol. 175, 2017

AtTTM1 Is Involved in Dark-Induced Leaf Senescence

AtTTM1 is an important (integral) part of the senescence pathway. Further evidence for this comes from several transcriptome studies (Lin and Wu, 2004; Buchanan-Wollaston et al., 2005). Buchanan-Wollaston et al. (2005) compared the transcriptional response of natural senescence with dark-induced senescence. They found that 827 genes were upregulated in senescent leaves compared to nonsenescent leaves. Of these genes, approximately 53% were also upregulated in dark-induced senescent leaves. Many of these genes either have putative or determined roles in macromolecule degradation, carbohydrate metabolism, membrane transport, secondary metabolism, and autophagy. This suggests that there are clear molecular differences between natural and dark-induced senescence; however, the overlapping genes may constitute a core senescence pathway of components required for the execution of senescence (van der Graaff et al., 2006). AtTTM1 is upregulated in both data sets, and we observed a delayed onset of natural senescence in the ttm1 mutant, indicating that it likely plays a central role in the senescence program. Interestingly, AtTTM1 was also upregulated in another data set, which identified genes that are induced during starvation-induced senescence of suspension cells (Swidzinski et al., 2002). These cells also exhibited clear symptoms of PCD. AtTTM1 is part of a group of 229 genes that are upregulated in all three types of senescence, suggesting that AtTTM1 is part of a core senescence pathway of components required for the execution of senescence (van der Graaff et al., 2006). Here, we connect both AtTTM1 and AtTTM2 to PCD during senescence and pathogen-induced HR, respectively. Their precise molecular role(s) during these processes are not clear yet, but their mitochondrial localization is likely crucial for their biological function, since TTM1 DTM, which does not localize to the mitochondria, lost its ability to complement the ttm1 mutant phenotype. The involvement of mitochondria in plant PCD 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 transmembrane domains (Fig. 7C; Kriechbaumer et al., 2009) and our confocal analyses, we conclude that AtTTM1 and 2 are mitochondria-localized, tail-anchored proteins. These proteins are usually localized in the mitochondrial

Figure 8. AtTTM1 and AtTTM2 can complement each other. A, Total chlorophyll content was measured in Arabidopsis accession Columbia (Col) wild type, ttm1-1, and three independent ttm1-1 complementation lines expressing AtTTM1 under its native promoter (pTTM1:TTM1) or AtTTM2 under the TTM1 promoter (pTTM1:TTM2) 0 and 7 d after dark treatment of detached leaves. Each bar represents the mean 6 SE Plant Physiol. Vol. 175, 2017

(n = 3). Bars marked with different letters indicate a significant difference (P , 0.05). The experiment was repeated four times with similar results. B, Quantitative real-time PCR analysis of AtTTM1 and AtTTM2 expression in detached leaves of 4- to 5-week-old transgenic plants 0 and 7 d after dark treatment. Transcripts were normalized to AtEF1a. Each bar represents the mean 6 SE (n = 3). An asterisk indicates significant differences to day 0, Student’s t test, p , 0.01). C and D, Transient overexpression of CaMV35S:YFP-AtTTM1 and CaMV35S:YFP-AtTTM2 induces cell death in N. benthamiana 7 d after infiltration of A. tumefaciens 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. 481

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outer membrane with their catalytic domain facing toward the cytosol (Abell and Mullen, 2011). We could not unequivocally confirm the mitochondrial localization of AtTTM2 at the same resolution as AtTTM1 due to low expression levels of AtTTM2 (under its native promoter), but the facts that (1) it was also predicted to be a tailanchored protein (Marty et al., 2014; Fig. 7C) and (2) our analysis of transiently expressed AtTTM2 (under the strong CaMV35S promoter) showed an identical punctate pattern (Supplemental Fig. S9) strongly suggest that AtTTM2 is also a mitochondrial protein as AtTTM1. Furthermore, the fact that overexpression of either AtTTM1 or 2 induced senescence-like cell death in the dark supports our conclusion that AtTTM1 is involved in senescence-associated cell death and both proteins have the same or very similar enzymatic activity. A possible function of AtTTM1 could be in pyrimidine catabolism through its uridine kinase-like domain. Stasolla et al. (2004) suggested that decreased salvage of uracil and uridine and increased salvage of thymidine represent a metabolic switch for the induction of programmed cell death (PCD). However, recombinantly expressed AtTTM1 (and 2) did not display uridine kinase activity, suggesting a different role for AtTTM1. Furthermore, Arabidopsis encodes five UK/uracil phosphoribosyl transferase genes (AtUKL1–5), which can form uridine monophosphate from uracil or uridine (Islam et al., 2007). Since we confirmed the uridine kinase activity of AtUKL1 in this study, it is likely that AtTTM1 acts on some other substrate. Both AtTTM1 and 2 displayed low activity on ATP, ADP, or PPPi substrates, while high affinity was observed for PPi with a Km of 17 mM, which is comparable to the Km of AtTTM3 or NeuTTM for PPPi (43 mM and 21 mM, respectively; Moeder et al., 2013; Delvaux et al., 2011). We tested several biologically relevant diphosphates such as thiamine diphosphate (Jordan, 2007), ADP ribose (Adams-Phillips et al., 2010) and NADH (Ishikawa et al., 2010), all of which were not hydrolyzed by AtTTM1 and AtTTM2 (Ung and Yoshioka, unpublished data). Thus, it remains to be determined whether PPi is the biological substrate and whether there are other diphosphate substrates for AtTTM1 and AtTTM2. Both proteins possess a proper P-loop kinase domain with Walker A, Walker B, and lid domains (Leipe et al., 2003). The CYTH domain, on the other hand, lacks the signature EXEXK domain (TYILK). Furthermore, a number of conserved basic and acidic residues facing into the b-barrel are not conserved. These features are also conserved in TTM1/2 orthologs in other plant species, indicating that they may be relevant for their catalytic activity. AtTTM1 and 2 are the first TTM proteins that have been reported to possess activity for PPi, where TTM proteins have only been reported to exclusively catalyze the hydrolysis of triphosphate compounds (Bettendorff and Wins, 2013). Iyer and Aravind (2002) suggested that the altered CYTH domain in AtTTM1/2 and their ortholog in D. discoideum may have lost its catalytic activity and serves as an allosteric binding site to modify the activity of the kinase 482

domain. Alternatively, the CYTH domain may serve as a binding site for a substrate that is phosphorylated by the P-loop kinase domain. PPi is the byproduct of a variety of biosynthetic reactions. Removal of PPi prevents these reactions from reaching equilibrium and plays an important role in maintaining the direction of these reactions (Maeshima, 2000). Arabidopsis encodes six soluble inorganic PPases and three membrane-bound H+-translocating PPases (Schulze et al., 2004; Ferjani et al., 2011). The H+ translocating PPases are located in the tonoplast or the Golgi apparatus, while the soluble PPases are located in the cytosol or chloroplast (AtPPa6; Schulze et al., 2004). None of these genes are up-regulated in senescing leaves like AtTTM1. Therefore, it is possible that AtTTM1 might specifically contribute to maintain low PPi levels during senescence. Its function could be to remove PPi, which may be necessary to drive a reaction at the mitochondrial outer membrane. Further analysis of ATTM1’s molecular mechanism is in progress. ttm1 and ttm2 knockout lines displayed distinct phenotypes in senescence and disease resistance, respectively. Furthermore, phylogenetic analysis indicates that most dicotyledonous plants maintain TTM1 and TTM2 paralogs (Supplemental Fig. S1) that fall into separate clades, further supporting the notion of distinct roles of these genes in plants. However, considering the high homology in their catalytic region (98% and 91% similarity in the uridine kinase and CYTH domains, respectively), it was of question whether they would act on the same in vivo substrate. Therefore, we tested whether AtTTM2, under control of the AtTTM1 promoter, could complement the ttm1 mutant phenotype and vice versa. Both paralogs could clearly complement the ttm1 mutant phenotype, while both constructs under control of the AtTTM2 promoter only exhibited a partial rescue. This is probably due to the intricate nature of the Hpa infection assay. Another explanation could be that the cDNA clones used lack some regulatory element compared to a genomic clone. This is supported by the fact that the pTTM2:TTM2 construct also complemented poorly. However, our data from the promoter swap experiments and the fact that overexpression of both AtTTM1 and 2 in N. benthamiana caused a cell death phenotype in the dark strongly suggest that the in vivo substrate is likely similar or identical for both proteins. At this time, we cannot explain why these two genes can complement each other yet their mutant phenotypes are distinctly different. Further studies into their biochemical properties may help to answer this question. It is well known that autophagy plays an important role in both pathogen-induced PCD and senescence (Hofius et al., 2017; Yoshimoto et al., 2009). Autophagy can play prosurvival or prodeath roles (Hofius et al., 2017), similar to what we see for AtTTM2 and AtTTM1, respectively. Potential roles for AtTTM1 and 2 in autophagy may explain the different functions in senescence and pathogen resistance. Further studies Plant Physiol. Vol. 175, 2017

AtTTM1 Is Involved in Dark-Induced Leaf Senescence

on a possible role of AtTTM1 and 2 in autophagy are underway. In summary, we present evidence suggesting that AtTTM1 and AtTTM2 have distinctive biological roles and that the spatial and temporal differences in their expression determine the different functions during senescence and disease resistance, respectively. With this study, we completed the basic characterization of all three Arabidopsis TTMs. These proteins exist in all domains of life, yet the biological role of most family members is not clear, and until recently there was no information available about their role in plants. Thus, we have laid the groundwork for the further study of plant TTM genes. Elucidation of the molecular mechanisms underlying the involvement of AtTTM1 and AtTTM2 in these processes is underway. MATERIALS AND METHODS Plant Growth Conditions and Pathogen Assays Arabidopsis (Arabidopsis thaliana) accession Columbia (Col) plants were grown in Sunshine Mix in a growth chamber at 22°C, 60% relative humidity, and ;140 mE m22 s21 with a 9-h photoperiod. Seven to ten-day-old Arabidopsis plants were infected with Hyaloperonospora arabidopsidis (Hpa). Spore counts of 8 3 105 cells mL21 and 2 3 105 cells mL21 were used for Emco5 and Emwa1 isolates, respectively. Seedlings were then infected via drop inoculation and left in a growth chamber at 16°C, .90% relative humidity for 7 to 10 d before disease 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 of Plant Breeding Research (Alonso et al., 2003; Kleinboelting et al., 2012), respectively. Homozygous plants (Arabidopsis accession Columbia) were isolated 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 the SALK line, LBb1-F, and the GABI-Kat line, GABIKAT-TDNA-F. Semiquantitative RT-PCR was then performed on cDNA from both ttm1 lines to confirm the 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 at the 1067-bp position (end of exon 3) and 2693-bp position (middle of exon 9) of ttm1-1 and ttm1-2, respectively (Supplemental Fig. S5). Primer sequences are listed in Supplemental Table S2.

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

RNA Extraction and RT-PCR RNA extraction was carried out using the TRIzol reagent (Life Technologies), according to the manufacturer’s instructions. Reverse transcriptase (RT)-PCR was performed using cDNA generated by SuperScript II Reverse Transcriptase (Life Technologies) according to the manufacturer’s instructions. Expression of PR1, CAB6, SAG12, and SAG13 was visualized by gel electrophoresis of samples after RT-PCR with the following RT primers: AtPR1-F, AtPR1-R, AtCAB6F, AtCAB6-R, AtSAG12-F, AtSAG12-R, AtSAG13-F, and AtSAG13-R. All primer sequences are listed in Supplemental Table S2.

Quantitative Real-Time PCR Quantitative real-time PCR was performed using Fast SYBR Green Master Mix (Life Technologies). The expression of Arabidopsis genes was normalized to the expression of AtEF1A (elongation factor1-alpha). All primer sequences are listed 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 plants were detached and floated on tap water in petri dishes in the dark for the specified 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 chlorophyll content was quantified by measuring the absorbance of chlorophylls A and B using a spectrophotometer and the equation, total chlorophyll content = 17.76 (A646) + 7.34 (A663) (Porra et al., 1989), followed by normalization to fresh weight.

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

Trypan Blue Staining

Promoter Swap Analysis

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

For the promoter swap analysis, full-length AtTTM1 or AtTTM2 cDNA with a C-terminal HA tag was cloned using TTM1swap-F and TTM1swap-R and TTM2swap-F and TTM2swap-R, respectively. The inserts were used to replace the uidA gene in the pORE R2 plant expression vector carrying the AtTTM1 or AtTTM2 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.

Confocal Microscopy For visualization of the mitochondria in Arabidopsis root cells, 4- to 10-d-old seedlings were soaked in 50 nM MitoTracker Orange (Invitrogen) dissolved in sterilized water for 1 min at 23°C. For FM4-64 staining, 4- tp 10-d-old seedlings were 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 roots were 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 the transition 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-Apochromat 633/1.40 oil immersion objective lens. For confocal microscopy of Nicotiana benthamiana leaves, 1-cm sections of Agrobacterium-infiltrated leaves were excised 24 h postinfiltration for Plant Physiol. Vol. 175, 2017

Agrobacterium-Mediated Transient Expression N. benthamiana was grown on Sunshine mix soil (Sun Gro Horticulture Canada) 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 483

Ung et al. in the figures) was infiltrated alone or coinfiltrated with constructs as described in 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-1 vector from Arabidopsis Columbia ecotype cDNA using the primers pGEXTTM1-F, pGEX-TTM1-R, pGEX-TTM2-F, and pGEX-TTM2-R, which exclude the annotated C-terminal transmembrane domain and end at S621 and D648 of AtTTM1 and AtTTM2, respectively. Each plasmid was introduced into the E. coli BL21 codon plus cells and grown overnight in Luria-Bertani medium at 37°C. The overnight culture was used to seed a larger volume of Autoinduction medium containing 13 NPS solution (25 mM (NH4)2SO4, 50 mM KH2PO4, and 50 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 temperature was then lowered to 18°C overnight before harvesting the cells by centrifugation at 4°C.

Protein Extraction E. coli cultures were centrifuged and pellets were resuspended in 13 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 fluoride, 1 mM dithiothreitol, and 10 mg mL21 DNase I. Cell suspensions were incubated on ice for 30 min before cell lysis by French press at 1000 psi. Soluble fractions were obtained by centrifugation and subjected to column purification using DE52 cellulose (Sigma) and GSH agarose (Sigma). Purified protein samples were eluted using 10 mM reduced glutathione, quantified using Bradford reagent, and stored at 280°C until use.

Malachite Green Assay Detection of free phosphates was performed as previously described (Bernal et al., 2005; Moeder et al., 2013).

Statistical Analysis A two-tailed Student’s t test was performed for all comparisons between two sample 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 samples with multiple groups.

Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: 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 relationship of TTM family members. Supplemental Figure S2. Amino acid sequence alignment of AtTTM1 and AtTTM2. Supplemental Figure S3. AtTTM1 and AtTTM2 do not exhibit uridine kinase activity. Supplemental Figure S4. Expression patterns of AtTTM1 and AtTTM2. Supplemental Figure S5. T-DNA insertion line analysis. 484

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 apparatus or endosome. Supplemental Figure S9. Subcellular localization of YFP-AtTTM1 and YFP-AtTTM2. Supplemental Figure S10. Pathogen infection phenotype of promoter swap 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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