Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:119–132 DOI 10.1007/s00210-014-1009-x

REVIEW

Clues to the functions of plant NDPK isoforms Sonia Dorion & Jean Rivoal

Received: 26 April 2014 / Accepted: 15 June 2014 / Published online: 26 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract This review describes the five nucleoside diphosphate kinase (NDPK) genes found in both model plants Arabidopsis thaliana (thale cress) and Oryza sativa L. (rice). Phylogenetic and sequence analyses of these genes allow the definition of four types of NDPK isoforms with different predicted subcellular localization. These predictions are supported by experimental evidence for most NDPK types. Data mining also provides evidence for the existence of a novel NDPK type putatively localized in the endoplasmic reticulum. Phylogenic analyses indicate that plant types I, II, and III belong to the previously identified Nme group I whereas type IV belongs to Nme group II. Additional analysis of the literature offers clues supporting the idea that the various plant NDPK types have different functions. Hence, cytosolic type I NDPKs are involved in metabolism, growth, and stress responses. Type II NDPKs are localized in the chloroplast and mainly involved in photosynthetic development and oxidative stress management. Type III NDPKs have dual targeting to the mitochondria and the chloroplast and are principally involved in energy metabolism. The subcellular localization and precise function of the novel type IV NDPKs, however, will require further investigations. Keywords Plant . Nucleoside diphosphate kinase . Isoform . Stress . Metabolism . Growth and development Abbreviations AK Adenylate kinase ANT Adenine nucleotide translocator ER Endoplasmic reticulum GFP Green fluorescent protein S. Dorion : J. Rivoal (*) IRBV, Université de Montréal, 4101 rue Sherbrooke est, Montréal, QC H1X 2B2, Canada e-mail: [email protected]

IM IMS NDP NDPK NTP PCD ROS

Inner membrane Intermembrane space Nucleoside diphosphate Nucleoside diphosphate kinase Nucleoside triphosphate Programmed cell death Reactive oxygen species

Introduction Nucleoside diphosphate kinases (NDPKs, EC 2.7.4.6) are key enzymes ubiquitously found in all organisms from bacteria to human and show remarkable sequence conservation. They catalyze the transfer of the terminal phosphoryl group of a donor nucleoside triphosphate (NTP) to an acceptor nucleoside diphosphate (NDP) in the presence of divalent cations, preferably Mg2+. NDPKs have broad substrate specificity, can use both ribo- and deoxyribonucleotides of purines or pyrimidines (Parks and Agarwal 1973), and are not known to be allosterically regulated. In all organisms, NDPKs are considered as housekeeping enzymes involved in energy metabolism and homeostasis of intracellular NTP pools (Dancer et al. 1990; Lambeth et al. 1997; Roberts et al. 1997; Bernard et al. 2000). NDPKs are therefore major players in the synthesis of macromolecules since they provide the neosynthetized triphosphates used for cell anabolic processes. They supply NTPs for nucleic acid synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis, and GTP for protein elongation, signal transduction, and microtubule polymerization. In various systems, NDPKs also participate in a wide variety of processes through mechanisms that are not necessarily connected to nucleoside phosphorylation (Boissan et al. 2009; Hsu 2011; Marino et al. 2012).

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The first plant NDPK was purified from pea seed in 1971 (Edlund 1971), and the first plant NDPK sequence was published 20 years after (Nomura et al. 1992). In the light of recent advances made using transcriptomic and proteomic approaches, it is now established that all plants contain a small NDPK gene family. So far,five annotated NDPK genes are found in the genomes of the model plants Arabidopsis thaliana (thale cress) and Oryza sativa L. (rice). Similarly to other eukaryotes, the occurrence of NDPK isoforms in plants has been documented in various subcellular compartments (Nomura et al. 1991; Finan et al. 1992; White et al. 1993; Lubeck and Soll 1995; Zhang et al. 1995; Struglics and Hakansson 1999; Escobar Galvis et al. 1999). Subcellular localization is an important factor in functional specialization since it can dictate substrate access or availability of interacting partners. As a matter of fact, there is increasing evidence that different plant NDPK isoforms participate in distinct processes besides their crucial role in NTP metabolism. Recent progress corroborates the idea that plant NDPK isoforms are also multifunctional proteins involved in development, stress responses, cell signaling, and coordination of other proteins. In this review, we analyze the current knowledge on plant NDPKs. According to the current official gene nomenclature, NDPKs belong to the Nme protein family, and a classification of animal Nme protein into two strongly supported clusters (groups I and II) has been proposed (Desvignes et al. 2009, 2010). Here, we first examine the composition of the NDPK gene family of two model plants representing the main flowering plant families: A. thaliana, a dicotyledon and rice, a monocotyledon. Phylogenic analyses indicate that plant NDPKs are represented in the two previously identified goups of Nme proteins. We present the different NDPK proteins encoded by these genes and review their subcellular localization using prediction programs and published evidence. According to their subcellular localization and their phylogenic relationships, we present the organization of the plant NDPK family in four distinct types, including a novel one discovered through data mining. Furthermore, we address the question of the functional specialization for the different plant NDPK isoforms by analyzing expression and biochemical studies as well as transgenic data from the literature. Except in the case of the novel type IV NDPK, we have restricted our review to the reports where the isoform was clearly identified.

Analysis of the NDPK gene families of A. thaliana and rice allows the definition of four types of NDPK isoforms in plants To understand the different plant NDPK isoforms, it is important to first review the composition of NDPK gene family. For

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this, we used data mining to retrieve the expressed NDPK genes found in representative models of the two main groups of flowering plants. The genome of A. thaliana (dicotyledon group) contains five annotated NDPK genes (Table 1) with the following loci identification numbers: At4g09320, At5g63310, At4g11010, At4g23900, and At1g17410 and, respectively, encoding AtNDPK1 to AtNDPK5 isoforms. Another locus, At4g23895, is annotated as encoding an NDPK protein in A. thaliana. The gene product is predicted to be a fusion between a protein containing a pleckstrin homology domain at the N-terminus and the sequence of NDPK4 at the C-terminus. However, up to date, there is no evidence for the expression of this fusion protein in the Arabidopsis database. The coding sequence corresponding to At4g09320 also appears to be erroneously annotated given data from phylogenic and sequence analyses (see Fig. 1 below). Indeed, the sequence of AtNDPK1 should start with the consensus N-terminal motif MEQTFI found in dicotyledonous NDPK1s (Dorion et al. 2006b). Among the A. thaliana genes, NDPK encoded by At1g17410 appears to not have been reported before in the literature, but there is evidence for its expression. In rice (monocotyledon group), five expressed NDPK genes are also known (Table 1) with the following loci identification numbers: Os07g0492000, Os12g0548300, Os05g0595400, Os10g0563700, and Os02g0565100 and encoding, respectively, OsNDPK1 to OsNDPK5 isoforms. Among these, to our knowledge, the latter has not been previously reported in the literature. Figure 1 presents the alignments of A. thaliana and rice predicted NDPK protein sequences. All sequences share a catalytic His residue which autophosphorylates as part of the catalytic mechanism (Lascu and Gonin 2000). Several sequences exhibit an N-terminal extension supporting organellar targeting (see below). Plant NDPKs have also been reported to autophosphorylate on two conserved Ser residues. The first is found immediately downstream of the catalytic His and appears necessary for full activity (Dorion et al. 2006a). In this case, phosphorylation is probably due to an intermolecular phosphotransfer (Dar and Chakraborti 2010). Using the pea ortholog of AtNDPK3, autophosphorylation of another strictly conserved Ser residue located at position 155 of the AtNDPK3 sequence was reported (Johansson et al. 2004). This residue was also shown to be necessary for the stability of NDPK oligomeric state and full activity of the enzyme (Johansson et al. 2004). Further analysis of the NDPK protein sequences reveals the presence of an endoplasmic reticulum (ER) retention signal (HDEL) at the C-terminal of the newly identified AtNDPK5 and OsNDPK5. The sequences were also analyzed using various subcellular targeting prediction programs (Table 1). Up to recently, plant NDPK isoforms were classified in three

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Table 1 Classification and predicted subcellular localization of NDPK isoforms found in A. thaliana and rice Type

I

Locus identifier

II

At4G09320 Os07g0492000 (LOC_Os07g30970.1) Os10g0563700 (LOC_Os10g41410.2) AT5G63310

III

Os12g0548300 (LOC_Os12g36194.1) AT4G11010 AT4G23900

IV

Os05g0595400 (LOC_Os05g51700.1) AT1G17410 Os02g0565100 (LOC_Os02g35700.1)

Isoform

AtNDPK1 OsNDPK1 OsNDPK4 AtNDPK2 (AtNDPK1a) OsNDPK2 AtNDPK3 (AtNDPK3a) AtNDPK4 (AtNDPK3b) OsNDPK3 AtNDPK5 OsNDPK5

Subcellular localization prediction WoLF PSORT

Predotar

TargetP

cytosol cytosol cytosol plastid

not organellar not organellar not organellar plastid

not organellar not organellar not organellar plastid

plastid mitochondria

plastid plastid

plastid plastid

mitochondria

plastid

mitochondria

mitochondria plastid extracellular

not organellar endoplasmic reticulum endoplasmic reticulum

plastid secretory pathway secretory pathway

Loci and isoform identifiers correspond to NCBI accession numbers or to denominations found the rice genome annotation project (http://rice. plantbiology.msu.edu/) or in the Arabidopsis information resource (http://www.arabidopsis.org/). Alternate denominations appear in parenthesis. Subcellular localization predictions were done using the protein sequence of each isoform and the programs available at the following websites: WoLF PSORT (http://wolfpsort.seq.cbrc.jp); Predotar (http://www.inra.fr/Internet/Produits/Predotar), TargetP (http://www.cbs.dtu.dk/services/TargetP)

distinct types (types I–III) that matched the subcellular localization of the proteins (Hammargren et al. 2007b). Type I NDPKs lack an identifiable targeting sequence and are therefore thought to be located in the cytosol (Dorion et al. 2006b), while types II and III NDPKs have Nterminal extensions that suggest targeting of the protein to the chloroplast and/or mitochondria (Sweetlove et al. 2001). However, given the retrieval of the AtNDPK5 and OsNDPK5 genes, we propose the addition of type IV NDPKs putatively localized in the ER to the plant NDPK family portrait. We performed a phylogenic analysis of the plant protein sequences together with a large selection of proteins from the animal Nme family using Clustal Omega (Fig. 2a). The plant protein sequences were also analyzed separately (Fig. 2b). According to the most recent classifications (Desvignes et al. 2009, 2010), the Nme protein family has been divided into two evolutionary distinct groups. Group I contains Nme1, Nme2, Nme3, Nme4, Nme-LV, and NmeGp1 whereas group II contains Nme5, Nme6, Nme7, Nme8, and Nme9. Recently evolved Nme10 proteins only possess a partial NDPK domain which makes them distinct from groups I and II (Desvignes et al. 2009). Figure 2a indicates that plant types I–III are part of group I. The phylogenic analysis positions them close to the Nme3 and Nme4 branches in the tree. Type IV NDPKs cluster with group II and are more closely related to the Nme5 group. Interestingly, Nme5 proteins appear to lack NDPK activity (Desvignes

et al. 2009). Whether Type IV NDPKs are active or not remains to be investigated. Type I NDPKs are predicted to localize predominantly in the cytosol Phylogenic data and prediction of subcellular localization support the grouping of AtNDPK1, OsNDPK1, and OsNDPK4 in type I NDPKs (Fig. 2, Table 1). Hence, rice contains two predicted cytosolic NDPKs. The predicted subcellular localization of type I NDPKs in the cytosol (Table 1) is supported by experimental studies using transient expression of AtNDPK1 and OsNDPK1 fused to fluorescent proteins (Reumann et al. 2009; Kihara et al. 2011). Interestingly, AtNDPK1 was also reported to partially localize to the nucleus and the peroxisome despite the absence of appropriate computationally predictable targeting signal mechanisms (Reumann et al. 2009). It was suggested that the protein could be either surface associated with or imported into peroxisomes via alternative yet unknown import mechanisms (Reumann et al. 2009). This is reminiscent of a similar situation described for Nme23/NDPK A (NME1) and Nme23/NDPK B (NME2), which also belong to group I. These proteins, while having a predominantly cytoplasmic localization, were shown to be occasionally present in the nucleus despite a lack of a nuclear localization signal (Bosnar et al. 2009). The cytosolic localization of type I NDPKs may thus not be exclusive. However, factors involved in

122 Fig. 1 Alignment of deduced amino acid sequences of NDPK genes found in A. thaliana and rice. Sequences were aligned using CLUSTAL OMEGA available at https://www.ebi.ac. uk/Tools/msa/clustalo/. Conserved amino acids are marked by asterisk. Similar amino acid residues with strongly or weakly similar properties are, respectively, identified by the symbols (:) and (.). Some sequence features discussed in the text are identified. The consensus N-terminal sequence found in dicotyledonous type I NDPK1 (MEQTFI) is underlined. The catalytic His residue is shaded in black. Phosphorylated Ser necessary for the stability of NDPK oligomeric state is in bold. The phosphorylated Ser residue downstream of the catalytic His appears in bold shaded gray. The HDEL ER retention signal identified at the C-terminus of type IV NDPKs is in bold. The numbering on the right refers to each sequence

Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:119–132 AtNDPK1 OsNDPK1 OsNDPK4 AtNDPK2 OsNDPK2 AtNDPK3 AtNDPK4 OsNDPK3 AtNDPK5 OsNDPK5

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------MVGATVVSKWTPLCVASPPERNSASLNPH------CSPARVNFRTALAAFRPQFRLFSRN ---------MDAMAVLARTSRPAPTLLAATSPAVSRRPAAVSFAAAAS---P---------------MSSQICRSA--SKAAKSLLSS----------------AKN-----ARFFSEG --------MSSQICRSA--SRAARSLLSS----------------AKN-----ARFFSEG ---------MSKLCQSA--CKAAKSLLSA----------------TAAASSPRTSLLAEG -----------------------------------------------------------------------------------------------------------------------

0 0 0 54 40 29 29 33 0 0

AtNDPK1 OsNDPK1 OsNDPK4 AtNDPK2 OsNDPK2 AtNDPK3 AtNDPK4 OsNDPK3 AtNDPK5 OsNDPK5

-----------------------------------------------------------M -----------------------------------------------------------M ---------------------------------------------------------MAL S----------------------ASRRRLRAS----SS----A-ESGIFLPHLVASMEDV -----------------------GSRGRVALSAAWGGR----A-ARGRVSAAGRIVASSV RAIGAAAAVSASGKIPLYASNFARSSGSGVASKSWITGLLALP-AAAYMIQDQEVLAAEM RAIGAASVVHATGKVPQYASNFGK-SGSGFVSNSWITGLLALP-AAAFMLQDQEALAAEM RNAALATLTNLGRKTLPTAYAYSYHHNSSAAAAGWL---AAIP-AAVYMLQDQEAHAAEM ----------MSG--------------ITYQ---IL-FLLLLASVSLSPVRCLGYGASSE ----------MGG--------------ATSPAAPCL-SVCLLPLLFLFLHGCWSCVAIER

1 1 3 83 72 88 87 89 32 35

AtNDPK1 OsNDPK1 OsNDPK4 AtNDPK2 OsNDPK2 AtNDPK3 AtNDPK4 OsNDPK3 AtNDPK5 OsNDPK5

EQTFIMIKPDGVQRGLIGEVICRFEKKGFTLKGLKLISVERSFAEKHYEDLSSKSFFSGL EQSFIMIKPDGVQRGLIGDIISRFEKKGFYLRGMKFMNVERSFAQQHYADLSDKPFFPGL EQTFIMIKPDGVQRGLIGEVIGRFEKKGFYLKAMKLINVEKSFAEKHYADLSSKPFFGGL EETYIMVKPDGIQRGLVGEIISRFEKKGFKLIGLKMFQCPKELAEEHYKDLSAKSFFPNL EQSYIMIKPDGVQRGLVGEIISRFEKKGFVLKGLKLFQCPKDLAQEHYKDLKEKPFFPGL ERTFIAIKPDGVQRGLISEIISRFERKGFKLVGIKVIVPSKDFAQKHYHDLKERPFFNGL ERTFIAIKPDGVQRGLISEIITRFERKGYKLVGIKVMVPSKGFAQKHYHDLKERPFFNGL ERTFIAIKPDGVQRGLISEILSRFERKGFKLVAIKLVVPSKEFAQKHYHDLKDRPFFNGL ERTLAMIKPDGVSGNYTEEIKTIVVEAGFNIVKEMLTQLDKETASAFYEEHSSRSFFPHL ERTLAMIKPDGLSGNYTERIKEVILESGFDIVKEAVVQLDAERASLFYAEHSGRSFFDSL *.: :****:. : . . *: : . *. .* : . : ** *

61 61 63 143 132 148 147 149 92 95

AtNDPK1 OsNDPK1 OsNDPK4 AtNDPK2 OsNDPK2 AtNDPK3 AtNDPK4 OsNDPK3 AtNDPK5 OsNDPK5

VDYIVSGPVVAMIWEGKNVVLTGRKIIGATNPAA---SEPGTIRGDFAIDIGRNVIHGSD VEYIISGPVVAMVWEGKDVVATGRRIIGATRPWE---AAPGTIRADYAVEVGRNVIHGSD VEYIVSGPVVAMVWEGKQVVSTGRKLVGATNPLA---AEPGTIRGDFAVDIGRNVIHGSD IEYITSGPVVCMAWEGVGVVASARKLIGKTDPLQ---AEPGTIRGDLAVQTGRNIVHGSD IEYITSGPVVCMAWEGNGVVASARKLIGATNPLQ---AEPGTIRGDLAVQTGRNVVHGSD CDFLSSGPVIAMVWEGDGVIRYGRKLIGATDPQK---SEPGTIRGDLAVTVGRNIIHGSD CNFLSSGPVVAMVWEGEGVIRYGRKLIGATDPQK---SEPGTIRGDLAVVVGRNIIHGSD CDFLSSGPVLAMVWEGEGVIKYGRKLIGATDPQK---SEPGTIRGDLAVVVGRNIIHGSD VTYMTSGPVLVMVLEKRNAVSDWRDLIGPTDAEKAKISHPHSIRALCGKNSQKNCVHGSD VKYMTSGPVLVMILERPDAISHWRVLIGPTDARKAKISNPNSIRAMCGVDSEKNCVHGSD :: ****: * * .: * ::* * : * :**. . :* :****

118 118 120 200 189 205 204 206 152 155

AtNDPK1 OsNDPK1 OsNDPK4 AtNDPK2 OsNDPK2 AtNDPK3 AtNDPK4 OsNDPK3 AtNDPK5 OsNDPK5

SVESARKEIALWFPDG-PVNWQSSVHPWVYETSVDNGKKEIALWFPEG-LAEWRSNLHPWIYESSVENARKEIALWFPEG-IAEWRSNQHPWIYEVSPENGKREIGLWFKEGELCKWDSALATWLRE-SPDNGKREIALWFKEGELCEWESVLTPWLVE-GPETAKDEISLWFKPQELVSYTSNSEKWLYGDN GPETAKDEISLWFKPEELVSYTSNAEKWIYGQN GPETAKAEIGLWFEPRELVSYTSNEEKWIYGVN STSSAEREIKFFFKDVVSGDIATQQHDEL---SPQSAAREISFFFGDVRSDTVE---HDEL----

alternate localization of these proteins remain unknown at this time. Type II NDPKs are chloroplastic proteins after all A. thaliana and rice each contain one representative of type II NDPKs, namely, AtNDPK2 and OsNDPK2 (Fig. 2, Table 1). Analysis of type II NDPKs using subcellular localization prediction tools indicates a plastidial localization (Table 1). Lubeck and Soll were the first to purify type II NDPK (NDPK2) from Pisum sativum L. (pea) chloroplasts (Lubeck

149 149 151 231 220 238 237 239 181 181

and Soll 1995). They showed that pea NDPK2 comprises two active forms of different sizes (18.5 and 17.4 kDa) within the chloroplast. The corresponding cDNA was cloned. By conducting in vitro import studies into isolated chloroplasts and in vivo analysis of transformed protoplasts, it was shown that the two mature forms of pea NDPK2 originate from a single mRNA, reside in the chloroplast stroma, and probably derive from specific proteolytic cleavage by a plastidic protease during organellar importation (Sharma et al. 2007). An 18kDa protein from Spinacia oleracea L. (spinach) chloroplasts was also purified to homogeneity using affinity

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Fig. 2 Phylogenic analyses of animal and plant Nme/NDPK proteins. Sequences were aligned as described in Fig. 1, and the phylogenetic tree was visualized using the TreeDyn software (Dereeper et al. 2008) available at http://www.phylogeny.fr/. a Phylogenic analysis of plant and animal protein sequences. The NCBI accession numbers are given in parenthesis, and the designation of the animal sequence clusters is done according to Desvignes et al. 2009. Group II cluster is highlighted by a dark gray background. Group I is shown using a light gray background. Nme10 cluster is on a white background. The various plant NDPKs are grouped by type and are highlighted by a green elliptical background. b Phylogenic analysis of A. thaliana and rice NDPK protein sequences. The different plant NDPK types are further discussed in the text

chromatography and identified as a type II NDPK (NDPK2) according to its N-terminal sequence (Yang and Lamppa 1996; Bovet et al. 1999). Surprisingly, in view of these data, AtNDPK2 was subsequently reported to have exclusive nuclear and cytoplasmic localizations in Arabidopsis (Zimmermann et al. 1999; Choi et al. 1999). The localization of AtNDPK2 was determined using a fusion of AtNDPK2 and the green fluorescent protein (GFP) transformed in Arabidopsis protoplasts (Zimmermann et al. 1999) and by examining leaf epidermal peelings of transgenic tobacco (Choi et al. 1999). However, a later report thoroughly revisited the localization of AtNDPK2 (Bölter et al. 2007). By using diverse methods such as in vitro importation, in vivo localization of GFP fusion proteins in A. thaliana transformed protoplasts, and immunoblotting of plant cell fractions, it

was unambiguously demonstrated that AtNDPK2 was exclusively targeted to chloroplasts where it colocalized with nucleoids. AtNDPK2 has a predicted chloroplast transit peptide at its N-terminus which is removed upon organellar import. AtNDPK2-GFP fusion protein, when transformed into isolated protoplasts, was expressed in chloroplasts whereas the protein with a deleted transit peptide localized to the cytosol. These results clearly indicate that the N-terminal extension of AtNDPK2 is necessary and sufficient for targeting to chloroplasts. These results were further validated by biolistic transfection of epidermal pavement cells of Bellis perennis L. (lawn daisy) with AtNDPK2-GFP (Jaedicke et al. 2011). These cells are ideal for the study of protein targeting to plastid because their pavement cells have chloroplasts, unlike almost all other epidermal pavement cells (including those of

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Arabidopsis, tobacco, and onion) which only bear proplastids (Jaedicke et al. 2011). Again, an inhomogeneous fluorescence signal likely associated with binding of AtNDPK2-GFP to chloroplast nucleoids was detected. In addition, a study of the proteome of A. thaliana chloroplast also reported a significant level of AtNDPK2 in the chloroplast (Peltier et al. 2006). Thus, the previously reported localization of AtNDPK2 in the nucleus and cytoplasm is now considered to be likely an artifact resulting from the use of inadequate constructs for establishing the location of AtNDPK2 (Bölter et al. 2007; Jaedicke et al. 2011). Indeed, Choi et al. (1999) reported fusing the GFP to the atNDPK2-Δ1–79 sequence, which lacked the transit peptide, whereas Zimmermann et al. (1999) mentioned fusing the full length of AtNDPK2 and a construct without the transit peptide (AtNDPK2-Δ1–79) to the C-terminus of the GFP thereby rendering the AtNDPK2 transit peptide inoperative for translocation of the protein into chloroplasts. Consistently with this, expression of GFP alone yields cytoplasmic and nuclear fluorescence in the transfected cell (Jaedicke et al. 2011). Type III NDPKs appear dually targeted to the mitochondria and the chloroplast The third NDPK type defined by phylogenic analysis and prediction of subcellular localization contains AtNDPK3, AtNDPK4, and OsNDPK3 (Fig. 2, Table 1). Subcellular localization prediction programs indicate a more inconsistent targeting that points mainly toward mitochondria or plastid (Table 1). This variability is supported by experimental approaches. In a proteomic survey of plant mitochondria fractions, potato type III NDPK (NDPK3) and AtNDPK3 were first localized to the intermembrane space (IMS) of mitochondria (Sweetlove et al. 2001). Further investigations of submitochondrial fractions demonstrated that the pea type III NDPK (NDPK3) existed both as a soluble form in IMS and as a form strongly attached to the inner membrane (IM) of mitochondria (Knorpp et al. 2003). Using Western blotting with peptidespecific antibodies and chloroplast import studies, AtNDPK3 was also shown to be present in the thylakoid lumen of chloroplasts (Spetea et al. 2004). This dual localization of type III NDPKs to the mitochondria and the chloroplast was later supported by the results of transient expression in leaf protoplasts using an NDPK3-GFP fusion construct containing the full length precursor of NDPK3 from pea (Hammargren et al. 2007b). Moreover, Western blot analysis of subcellular fractions from pea further confirmed the dual localization of NDPK3 and demonstrated that the majority of the NDPK3 protein was found in the mitochondria (Hammargren et al. 2007b). More recently, using a GFP-fused protein in transient expression experiment, OsNDPK3 was exclusively targeted to mitochondria in onion epidermal cells (Kihara et al. 2011). However, these cells do not contain green plastids probably

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explaining the exclusive mitochondrial location (Spetea and Lundin 2012). The novel type IV NDPKs are predicted to be targeted to the ER AtNDPK5 and OsNDPK5 are clearly phylogenically related together and distinct from the other NDPK types (Fig. 2). Analysis of these sequences using subcellular localization prediction tools indicates a very likely targeting to the ER and the secretory pathway (Table 1). The only exception to this was the relatively weak prediction of a plastidial localization of AtNDPK5 by WoLF PSORT. This same analysis gave an ER and/or vacuolar localization as a non-negligible alternate possibility. In plants, the secretory pathway delivers proteins to the cell wall and apoplastic (extracellular) space, the vacuole, the tonoplast, and to the plasma membrane. Proteins that are directed to these compartments (secretory proteins) are cotranslationally inserted into the ER and are then sorted or targeted to and/or retained in various intracellular compartments. The predicted ER localization of type IV NDPKs is supported by the presence of the ER retention signal HDEL at the C-terminal. The H/KDEL signal is known to be sufficient for ER localization of soluble proteins (Vitale and Denecke 1999). It is also involved in the retrieval mechanism from the Golgi apparatus in order to maintain appropriate levels of proteins that are resident in the ER (Hanton et al. 2006). This H/KDEL motif is recognized by ERD2, a saturable receptor protein that cycles between the ER and the Golgi (Hanton et al. 2006). To our knowledge, no experimental data are yet available to support the predicted localization of type IV NDPKs.

Involvement of type I NDPKs in metabolism, growth and development, and stress responses Expression patterns of type I NDPKs point to a role in sugar nucleotide metabolism related to growth and development Gene expression analyses in Arabidopsis inflorescences, leaves, and roots using quantitative PCR showed that AtNDPK1 is the most highly expressed NDPK gene in all the studied tissues (Hammargren et al. 2007b). Consistently with these data, activity measurements, immunoblot analysis, and analytical purification of NDPK isoforms in vegetative and reproductive potato tissues also showed that type I NDPK is expressed in all tissues surveyed and constitutes the bulk of NDPK activity (70– 80 % of total extractable NDPK activity) (Dorion et al. 2006b). The cytosolic compartment of plant cells therefore contains the highest potential for NTP synthesis from

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ATP. In plants, a significant number of reports show that type I NDPKs can be spatially and temporally regulated during growth and development. Immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that type I NDPK expression was predominant in meristematic zones and provascular tissues of the apical regions (Dorion et al. 2006b). According to these results, it was suggested that this isoform could be more specifically involved in the supply of UTP for the synthesis of the precursors of the cell wall during early growth. Indeed, primary cell wall is normally laid out during cell division, and wall material deposition continues with the maturation of the cell. Provascular tissues are also sites of intense cell wall synthesis. The synthesis of precursors for the cell wall heavily depends on UTP which is required for the synthesis of the diverse family of UDP-sugars by UDP-sugar pyrophosphorylases (e.g., UDP-glucose pyrophosphorylase, EC 2.7.9.9) as well as for the synthesis of UDP-glucuronic acid by UDP-glucuronic acid pyrophosphorylase (EC 2.7.7.44) (Reiter and Vanzin 2001; Seifert 2004; McFarlane et al. 2014). During berry flesh development in Vitis vinifera L. (grapevine), from fruit set to the end of green fruit development, there is an increase in type I NDPK (NDPK1) expression and differential accumulation rates for its phosphorylated forms (Martinez-Esteso et al. 2011). The high levels of this protein in developing pericarp of grape berries could have a relevant function in berry growth in particular in primary cell wall synthesis (Martinez-Esteso et al. 2011), as previously suggested (Dorion et al. 2006b). Other evidence for such role comes from proteomic analyses of Quercus suber (cork oak) stem tissues which identified the presence of type I NDPK and UDPglucose pyrophosphorylase in the phellem (cork) but not in the xylem (Ricardo et al. 2011). Cork formation is associated with significant cell wall reorganization and biosynthesis of the major components of cork, including suberin, cellulose, hemicellulose, and lignin. Serial analysis of gene expression (SAGE) of the developing wheat (Triticum aestivum L.) caryopses uncovered abundant type I NDPK and cellulose synthase transcripts during early grain development (McIntosh et al. 2007). These data were later supported by proteomic characterization of developing wheat grain. High levels of type I NDPK expression were associated with the cell division and differentiation phase of grain development. Levels were gradually downregulated during subsequent grain filling and desiccation/maturation phases of grain development (Guo et al. 2012). A similar expression pattern was observed for OsNDPK1 during rice caryopsis development (Lin et al. 2005). The function of type I NDPK in growth and development is further supported of by the outcome of the transgenic manipulation of OsNDPK1 in rice (Pan

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et al. 2000). Expression of a heat-inducible antisense OsNDPK1 construct resulted in growth defect. Mature (60 days after sowing) transgenic plants were shorter than nontransformed plants. This phenotype suggests that OsNDPK1 is involved in the cell elongation processes as cell length was reduced in coleoptile epidermal cells of antisense plants (Pan et al. 2000). Involvement of type I NDPKs in stress responses A large number of publications have reported on the stress regulation of type I NDPKs. Upon exposure to salt stress, the transcript and/or the encoded protein were shown to be upregulated in roots of rice (Kawasaki et al. 2001) and pea (Kav et al. 2004), and in leaves of Hordeum spontaneum (wild barley) (Fatehi et al. 2013). Exposure of rice to cold temperatures was shown to induce leaf OsNDPK1 (Yang et al. 2006; Lee et al. 2007) and elevate the phosphorylation of root OsNDPK4 (Chen et al. 2012). Drought stress was also shown to induce the isoform in leaves of rice (Salekdeh et al. 2002), Beta vulgaris L. (sugar beet) (Hajheidari et al. 2005) and Arachis hypogaea L. (peanut) (Kottapalli et al. 2009). Type I NDPK expression was also increased by wounding in tomato leaf and stem (Harris et al. 1994). OsNDPK1 transcript was induced as well in rice leaves infected by the pathogen Xanthomonas oryzae (Cho et al. 2004). Type I NDPK expression was found to be upregulated in roots of Brassica napus (oilseed rape) in response to Plasmodiophora brassicae infection (Cao et al. 2008). Overexpression of AtNDPK1 was found to increase resistance to paraquat and confer a higher ability to eliminate exogenous H2O2 (Fukamatsu et al. 2003). This phenotype was linked to a possible interaction of AtNDPK1 with catalase (Fukamatsu et al. 2003). The explanation for enhanced expression under stress is far from being clear at the present time. In some cases, however, progress has been made on the mechanisms underlying this regulation. Induction under drought stress in Zea mays (maize) was shown to be relatively atypical since it was independent of abscissic acid signaling (Hu et al. 2011), whereas OsNDPK1 expression was shown to be stimulated by this hormone (Cho et al. 2004). OsNDPK1 was found to be positively regulated by salicylic acid and jasmonic acid, two hormones known to regulate the coordination of plant defenses against pathogens (Cho et al. 2004; Li et al. 2012). There is also evidence for increased phosphorylation of pea type I NDPK by red light irradiation (Tanaka et al. 1998). More recently, it was shown that expression of AtNDPK1 was negatively regulated by stress-induced MAP kinase kinase (SIMKK), which is involved in plant salt and pathogen sensing (Ovecka et al. 2014). These data indicate that stress regulation of type I NDPK results

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from a complex interplay of signaling by hormones and MAP kinase pathways.

Type II NDPK is associated with chloroplast function, oxidative stress, and auxin signaling The physiological relevance of protein-protein interactions detected in vitro can be questioned if the proteins are localized to different cell compartments in vivo. In the view of the reassessment of type II NDPK subcellular localization (see above), literature inferring a function for AtNDPK2 solely based on data from in vitro protein-protein interactions has to be carefully re-evaluated. For this reason, this section focuses on gene expression studies and literature data pointing to a function using mainly genetic approaches. Involvement in photosynthetic development In A. thaliana, NDPK2 transcript was found to be the highest in leaves and was also present in significant amounts in inflorescences but was low or absent in roots (Hammargren et al. 2007b; Verslues et al. 2007a). In Brassica campestris (chinese cabbage), expression of type II NDPK increased dramatically in seedlings transferred to light after dark germination (Shin et al. 2004). Furthermore, an A. thaliana ndpk2 knockout mutant showed a defect in red light-induced greening of the cotyledon compared to the wild type (Choi et al. 1999), linking AtNDPK2 to photosynthetic development. Thus, type II NDPK seems to be necessary for the proper establishment of photosynthetic function. Characterization of plants with altered type II NDPK levels point to a function in reactive oxygen species (ROS) signaling and oxidative stress management The ndpk2 knockout mutant had reduced root and seedling growth and higher levels of ROS and H2O2 compared to the wild type (Moon et al. 2003; Verslues et al. 2007b). Conversely, A. thaliana overexpressing AtNDPK2 had low ROS levels. Furthermore, resistance to methyl viologeninduced oxidative stress was positively correlated to AtNDPK2 expression level. Moreover, microarray analysis of A. thaliana constitutively overexpressing AtNDPK2 demonstrated induction of multiple antioxidant genes (Yang et al. 2003). Thus, type II NDPK’s protective role against oxidative stress makes it an interesting target for plant biotechnology. This stress-resistant phenotype was later observed when AtNDPK2 was overexpressed in Hordeum vulgare L. (barley) (Um et al. 2007), Solanum tuberosum (potato) (Tang et al. 2008), Ipomoea batatas L. (sweet potato) (Kim et al. 2009), and Populus (hybrid poplar) (Kim et al. 2011). A. thaliana ndpk2 characterization also showed that the

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mutant had increased Pro levels and altered response to abscissic acid. It was suggested that these were indirect effects of elevated H2O2 (Verslues et al. 2007b). Interestingly, AtNDPK2 was induced by H2O2 (Moon et al. 2003), thereby positioning it in a feedback loop where it is induced by H2O2 and serves to manage H2O2 levels. Further support for ROS regulation of type II NDPK came from analysis of transgenic tobacco BY-2 cell line with lowered ascorbate peroxidase expression which shows elevated ROS and upregulation of genes coding for ROS-scavenging enzymes, the MAP kinase kinase kinase NPK1 and type II NDPK (Ishikawa et al. 2005). ROS regulation of this NDPK isoform is relevant to its subcellular localization since photosynthesis is a source of ROS and H2O2 and because the chloroplast is involved in the triggering of ROS/redox-dependent signaling (Petrov and Van Breusegem 2012; Kangasjarvi et al. 2012). Pathogen attacks have also been linked to ROS production in the chloroplast. Indeed, pathogen infection was shown to activate the SIPK/Ntf4/WIPK MAPK kinase cascade, resulting in a sharp reduction of carbon fixation thereby promoting ROS production (Liu et al. 2007). Consistently with the above, OsNDPK2 was reportedly induced by a fungal elicitor in leaves (Liao et al. 2009).

Involvement in auxin-regulated processes The A. thaliana ndpk2 mutant displayed photomorphogenic defects that were linked to auxin regulation. Wild type and ndpk2 mutant relative hypocotyl length differences observed under red and far red light were attributed to regulation of phytochrome by direct interaction with AtNDPK2 (Choi et al. 1999, 2005). However, this idea was dismissed in a recent review on phytochromedependent light signaling (Hughes 2013). The exact role of type II NDPK in sensitivity to red/far red light therefore remains unclear at the moment. The ndpk2 mutant had increased sensitivity to an inhibitor of polar auxin transport and reduced transcript levels of several auxin responsive genes (Choi et al. 2005). Conversely, overexpression of AtNDPK2 in hybrid poplar increased expression of two auxin-regulated genes (Kim et al. 2011). Auxin transport was enhanced in ndpk2 compared to wild type. An interpretation of these results is that ndpk2 had modified levels of auxin due to an altered distribution (Choi et al. 2005). This was supported by abnormal gravitropic response of ndpk2 when auxin transport was inhibited (Choi et al. 2005). These effects of type II NDPK on auxin transport and sensitivity could perhaps be connected to the abovedescribed function of the isoform in ROS signaling. Indeed, ROS and auxin are interconnected in a complex signaling network involved in regulation of plant growth and development (Tognetti et al. 2012). It is therefore

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possible that the ndpk2 phenotype is the result of altered cross talk between ROS and auxin signaling pathways.

Developmental expression studies and identity of interaction partners of type III NDPKs point to a function in energy metabolism Expression data Several lines of evidence link type III NDPKs to energy metabolism. Treatment of A. thaliana leaves with sucrose or glucose, the substrates of respiratory metabolism, induced AtNDPK3 expression (Hammargren et al. 2008). It was hypothesized that this regulation was due to the presence in the promoter of AtNDPK3 of a binding site for the SUSIBA2 transcription factor which is involved in mediating sugar responses (Hammargren et al. 2008). The promoter of AtNDPK4 did not contain this feature, and AtNDPK4 mRNA expression was slightly repressed by sucrose (Hammargren et al. 2008). Rapidly dividing inflorescence and root tissues with high demands for mitochondrial activity showed high expression of the mitochondrial AtNDPK3 (Hammargren et al. 2007b). The expression of AtNDPK4 was estimated to be only about 10 % of that of AtNDPK3 (Hammargren et al. 2007b). Nonetheless, it is interesting to note that the transcript levels of AtNDPK4 are increased in inflorescence, a tissue with elevated energy demands and during later stages of flower development, specifically in the tapetum, ovules, and petals. It was hypothesized that AtNDPK4 can function as a complement to AtNDPK3 in tissues requiring high mitochondrial activity (Hammargren et al. 2007b). Northern blot analysis of type III NDPK performed at various developmental stages of pea leaves showed a higher expression in young leaves as compared to old ones (Escobar Galvis et al. 1999). It was suggested that the age-dependent decrease of transcript could be linked to the decline of mitochondrial respiratory activity once the leaf tissue becomes more photosynthetically active (Hammargren et al. 2007b). Type III NDPK interacts with other nucleotide-metabolizing enzymes The adenine nucleotide translocator (ANT) was identified as an interacting partner of the membrane bound pea type III NDPK in coimmunoprecipitation experiments (Knorpp et al. 2003). ANT is a mitochondrial IM localized carrier that mediates the exchange of cytosolic ADP for ATP synthesized in the matrix. Although NDPK catalyzes reversible phosphotransfer between various NDP and NTP

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species, permanent ATP delivery to the IMS via the ANT drives the phosphotransfer reaction toward the generation of other NTPs, principally GTP. In spinach, substrate specificities for NTPs and NDPs were shown to vary among NDPK isoforms. In particular, the Km value for GDP of type III NDPK was much lower than that for ADP, in contrast to observations made on other isoforms. This suggests a preferential involvement in the formation of GTP (Zhang et al. 1995). The function of this NDPKANT interaction could thus be to locally decrease the ATP concentration on the IMS side of the IM thereby facilitating a higher rate of ATP export from the matrix (Knorpp et al. 2003). Consequently, type III NDPK may also provide GTP in the direct vicinity of mitochondrial GTPases as in animals (Schlattner et al. 2013). Indeed, such function has been described for mammalian NDPK-D, the product of NME4 which is localized in the IMS where it is mainly bound to the IM (Milon et al. 2000; Tokarska-Schlattner et al. 2008). ADP regenerated in the IMS by the NDPK-D phosphotransfer reaction was shown to be directly taken up via ANT into the matrix space to stimulate respiratory ATP regeneration (Tokarska-Schlattner et al. 2008) and directly provide GTP for OPA1 (Optic Atrophy 1), a mitochondrial GTPase interacting with NDPK-D (Schlattner et al. 2013). NDPK-D binding to the IM occurs through electrostatic interaction between a canonic basic motif (Arg89‐Arg90‐ Lys91) and the negative charges of the polar head group of cardiolipin (Tokarska-Schlattner et al. 2008). Binding of plant type III NDPK to the IM is unlikely to occur through such a process since an acidic residue is present at the center of the triad motif preventing cardiolipin binding (Tokarska-Schlattner et al. 2008). The resistance of pea type III NDPK to washes with NaCO3 and Triton X-100 indicates that it is strongly attached to the inner membrane of mitochondria (Knorpp et al. 2003). This could indicate that mitochondrial NDPK, together with its interaction partner ANT, is localized to the contact points for channeling metabolites between the matrix and the cytoplasm (Knorpp et al. 2003). However, this possibility still needs to be confirmed experimentally. Interestingly, in chloroplasts, a homolog of the mitochondrial ANT, named the thylakoid ATP/ADP carrier (Thuswaldner et al. 2007), is present in the thylakoid membrane where it may analogously interact with the chloroplast lumen-localized type III NDPK (Spetea et al. 2004). Consequently, this isoform could also directly provide GTP for lumen GTPases. Requirement for GTP has been reported in thylakoid lumen for PsbO, a GTPase, mediating the turnover of the D1 subunit of photosystem II complex following inactivation induced by either high- or low-intensity light (Spetea et al. 1999, 2000). Adenylate Kinase (AK) was identified by affinity purification as an interacting partner of the soluble form of type III

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NDPK localized in the IMS of mitochondria (Johansson et al. 2008). AK participates in the homeostasis of adenylate pools in the plant cell (Hooks et al. 1994; Igamberdiev and Kleczkowski 2001). AK enzymes are ubiquitous and very abundant in organelles with high turnover of nucleotides, like chloroplasts and mitochondria, and have been shown to occur in the plant mitochondrial IMS (Igamberdiev and Kleczkowski 2001). It was hypothesized that mitochondrial NDPK could fine-tune the rates of catabolic versus anabolic processes, because ATP is produced by catabolism and GTP is typically consumed by anabolic reactions (Roberts et al. 1997). In vitro experiments using recombinant enzyme showed that NDPK and AK affected each other’s activity: AK stimulated NDPK activity whereas NDPK inhibited AK (Johansson et al. 2008). Nevertheless, the in vivo effects of the NDPK-AK interaction could be different, since the stoichiometry of these enzymes in the complex is still unknown. Interestingly, the oligomeric structure of NDPK is important for the inhibitory effect on AK activity, possibly for shielding the AK AMP binding site. Indeed, the activity of AK is unchanged when it interacts with the NDPK S69A mutant. This mutation prevents the formation of NDPK hexamers and results in only 5–10 % residual activity (Johansson et al. 2004). Intriguingly, salinity stress was shown to alter the ratio of the two proteins in a tissue-specific manner (Jacoby et al. 2013). These results raise the possibility that, under salinity stress, each tissue fine-tunes the abundance of nucleoside phosphates within the mitochondrial IMS in order to meet the specific metabolic and signaling requirements (Jacoby et al. 2013). The function of AK and NDPK in energy metabolism may be linked with programmed cell death (PCD) (Valenti et al. 2007). Mitochondria oxidative burst and outer membrane potential loss are early markers in A. thaliana PCD induced by various stimuli (Valenti et al. 2007). The energetic status of mitochondria is also important in the initialization of PCD. Indeed, ATP depletion has been reported in tobacco BY-2 cells and A. thaliana following PCD induction (Mlejnek et al. 2003; Tiwari et al. 2002). Mitochondrial NDPK was suggested to play a central part in the early phases of heat-shock-induced PCD in tobacco cells (Valenti et al. 2007). This hypothesis is interesting since, in the early phases of PCD, oxidative phosphorylation is impaired, coupling (i.e., ADP stimulation) of respiration is abolished and the ability of mitochondria to export ATP via ANT as well as the activities of both the AK and NDPK are inhibited without any decrease in protein levels. These events occur in a ROS-dependent manner (Valenti et al. 2007). In the light of these data, inhibition of type III NDPK by PCD could result in impairment of the regulation of adenylate pools and intracellular distribution. Moreover, by interacting with both the ANT and AK, inhibition of NDPK activity could also impair the function of these interactants.

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Fig. 3 Schematic representation of the predicted subcellular localization and recapitulation of the putative functions of the NDPK types found in plant cells as discussed in this review. CYT cytoplasm, CP chloroplast, ER endoplasmic reticulum, M mitochondria, N nucleus, P peroxisome

Some functions of type III NDPKs are still ill-defined A potential regulatory role for pea mitochondrial NDPK during heat stress was suggested (Escobar Galvis et al. 2001). In vivo labeling and coprecipitation assays showed that upon heat stress, the pea mitochondrial NDPK interacts with a newly synthesized 86-kDa protein of unknown function. Neither the steady-state levels of mitochondrial NDPK mRNA nor protein levels were regulated by heat stress. Functional implication of the interaction between NDPK and the 86-kDa protein still remains to be elucidated. Recombinant pea type III NDPK was reported to cleave DNA and RNA (Hammargren et al. 2007a). DNAbased activities of animal NDPKs (e.g. transcriptional

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regulation of c-myc or DNA cleavage) were abundantly reported but are now being re-evaluated because of the issue of the purity of recombinant NDPK preparations (Steeg et al. 2011). For the demonstration of the nuclease activity of plant NDPK, the purity of the recombinant protein was not assessed. Thus, the DNAse/RNAse activities attributed to the NDPK recombinant protein remains to be validated.

The enigmatic type IV isoform Data mining in the A. thaliana and the rice genomes revealed the existence of genes encoding the novel type IV NDPK, predicted to localize in the ER lumen. The characterization of AtNDPK5 and OsNDPK5 has not been carried out, so, it is not possible to assign a particular function to these proteins. However, it is tempting to make a connection between type IV NDPKs and scattered literature reports dating from the early days of plant NDPK studies on microsome-localized NDPK. A 17-kDa polypeptide isolated from pea microsomes was tentatively identified as NDPK based on limited N-terminal sequencing (Finan et al. 1992). However, this sequence was not discriminating enough to identify a particular NDPK type using the current GenBank database. Additional research on pea microsome NDPK showed that it was loosely associated with membranes (White et al. 1993). Western blot analysis of microsomes from the moss Physcomitrella patens also revealed the presence of an NDPK protein (Hutton et al. 1998). Similarly, a barley root microsomal fraction described to consist of ER, tonoplast, and plasma membranes was shown to contain a protein with the functional characteristics of NDPK which does not appear to have been further characterized (Reuveni and Dupont 2001). Additional reports of microsomal NDPK protein and activity come from work on A. thaliana and pea (Novikova et al. 1999, 2003). A. thaliana microsomal NDPK protein levels were unaffected in wild type and two ethylene insensitive mutants (eti5 and etr), whereas its phosphorylation level was elevated in the two mutants compared to the wild type (Novikova et al. 1999). This could be of interest since ethylene receptors are tethered to the ER membrane (Ju and Chang 2012). However, up to date, mutant screens have not yet identified NDPK as a component of the ethylene response pathway (Ju and Chang 2012; Merchante et al. 2013).

Conclusions and perspectives Figure 3 recapitulates the predicted subcellular localization of the various plant NDPK types, as well as the processes in

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which they are possibly involved, as discussed in this review. Much progress has been accomplished in our understanding of plant NDPKs. However, many questions still remain unanswered about this fascinating enzyme. We can identify a few interesting problems. What mechanisms are responsible for type I NDPKs regulation by a variety of stresses? How does type II NDPKs affect antioxidant gene expression? What is the specific involvement of type III NDPKs in PCD? What function(s) is linked to type IV NDPKs? Undoubtedly, the use of genetic approaches will help solve some of these issues in the coming years. Acknowledgments This work was supported by a Discovery Grant from the National Science and Engineering Research Council of Canada (NSERC) and a grant from the Projet de Recherche en Équipe from the Fonds de Recherche du Québec - Nature et technologies (FRQ-NT).

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