nucleotide metabolism in plants1[open] · nucleotide metabolism in plants1[open] claus-peter...

Update on Nucleotide Metabolism in Plants

Nucleotide Metabolism in Plants1[OPEN]

Claus-Peter Witte ,2,3 and Marco Herde

Leibniz Universität Hannover, Department of Molecular Nutrition and Biochemistry of Plants, HerrenhäuserStrasse 2, 30419 Hannover, Germany

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

NUCLEOTIDE METABOLISM IN PLANTS

Nucleotides are essential for life. It is easy to validatethis statement—one just needs to recall that nucleotidesare the building blocks of DNA and RNA, and thatmany molecules that are central for metabolism, forexample ATP, NADH, Co-A, and UDP-Glc, are nu-cleotides or contain nucleotide moieties. Generally, anucleotide is defined as a phosphorylated ribose ordeoxyribose linked to a nitrogen-containing hetero-cyclic group called the nucleobase via a glycosidicbond (Fig. 1). Because of the phosphate groups, nu-cleotides are negatively charged, whereas at neutralpH, nucleosides and nucleobases are uncharged. Theexception is xanthine, which is partially charged as afree base (pKa 7.4) but completely charged at the basein xanthosine (pKa 5.5) or the corresponding nucleo-tides (Fig. 1; Sigel et al., 2009).Many excellent reviews focus on general (Wagner

and Backer, 1992; Moffatt and Ashihara, 2002;Stasolla et al., 2003; Zrenner et al., 2006; Zrenner andAshihara, 2011) or particular aspects (Smith andAtkins, 2002; Kafer et al., 2004; Ashihara et al.,2018) of plant nucleotide metabolism. The aim ofthis review is to provide an update on how nucleo-tide metabolism is hardwired, mostly focusing onthe cellular level, because our understanding of theorganization at the tissue and organ level remainsvery limited. The presented models are mostly basedon results from Arabidopsis (Arabidopsis thaliana).These will often be valid for most plants, but cer-tainly there will be species-dependent variations.We also cover extracellular nucleotide metabolismand review the evidence for overlap between cyto-kinin metabolism and central nucleotide metabo-lism. Figure 2 shows a general overview of plantnucleotide metabolism.

DE NOVO SYNTHESIS

Purine De Novo Synthesis

Plants possess metabolic pathways for the de novosynthesis of purine nucleotides generating AMP, aswell as pyrimidine nucleotides yielding UMP. Duringde novo biosynthesis, nucleotides are newly synthe-sized from activated ribose (5-phosphoribosyl-1-py-rophosphate [PRPP]), Gln, Asp, and bicarbonate, aswell as specifically for the purine nucleotides Gly andformyl tetrahydrofolate (Fig. 2).There is strong evidence thatAMPbiosynthesis occurs

entirely in the plastids, because the 11 enzymes (cata-lyzing 12 reactions; Smith and Atkins, 2002) required forAMP biosynthesis in Arabidopsis all have anN-terminalorganelle-targeting peptide, and C-terminal yellow flu-orescent protein-fusion proteins of several of these en-zymes were observed exclusively in the plastids whenthey were transiently expressed inNicotiana benthamiana

1This work was supported by the Deutsche Forschungsgemein-schaft (WI3411/4-1 to C.-P.W. and HE 5949/3-1 to M.H.) and theBundesministerium für Bildung und Forschung (Nutzpflanzen derZukunft - 031B0540).

2Author for contact: [email protected] author.C.-P.W. conceived the study; C.-P.W. and M.H. wrote the article.[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00955

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in our laboratory (Fig. 3A; N. Medina Escobar andC.-P. Witte, unpublished data). In rice (Oryza sativa),the pathway also seems to reside in plastids (Zhanget al., 2018). However, it has been reported that innodules of the tropical legume cowpea (Vigna un-guiculata), purine biosynthesis is targeted to plastidsand mitochondria (Atkins et al., 1997; Smith andAtkins, 2002). It may be worthwhile to reconfirmthis special localization in nodules using fluorescenttagged proteins.

AMP is exported from the plastids by the adeninenucleotide uniporter BT1 (Fig. 3A, no. 1), which canalso transport ADP and ATP (Leroch et al., 2005;Kirchberger et al., 2008; Hu et al., 2017). Interestingly,BT1 from Arabidopsis and maize (Zea mays) wasreported to be dually localized to the chloroplast andmitochondria (Bahaji et al., 2011b) and a BT1 mutantwith a severe dwarf phenotype could be complementedwith a cDNA coding for an N-terminally truncatedversion of BT1 that is exclusively located in the mito-chondria and not in the plastids (Bahaji et al., 2011a).This either indicates that there are certain tissues or de-velopmental stages where purine nucleotide biosynthe-sis occurs mainly in mitochondria, or that BT1 has analternative and essential function in this organelle. In thelatter case, BT1 might still also be involved in exportingadenylates from plastids, but the abrogation of theplastidic variant would not cause the strong dwarfphenotype, indicating that de novo synthesized AMPhas an alternative, BT1-independent way to leave theplastids.

GMPbiosynthesis requires inosine 59-monophosphate(IMP; Fig. 3A), which can either be derived from AMPdeamination in the cytosolic compartment catalyzed byAMP deaminase (AMPD; Fig. 3A, no. 2) or from directexport of IMP from the plastids, because IMP is gen-erated there en route to AMP (Fig. 3A). Mutation ofAMPD is zygote lethal (Xu et al., 2005), and coformy-cin, an AMPD inhibitor, is a potent herbicide after itsphosphorylation in vivo (Dancer et al., 1997). Thesephenotypic effects might be caused by hampered GMPbiosynthesis, suggesting that AMPD could be requiredfor this process. Consistent with this, AMPD is stronglyactivated by ATP (Han et al., 2006), and this regulationmight balance cellular ATP and GTP concentrations.However, it has also been reported that AMPD inhibi-tion might be detrimental by severely altering the cel-lular energy charge and that the GTP pool is not alteredupon AMPD inhibition (Sabina et al., 2007), implyingthat GMP biosynthesis is independent of AMPD andthat there is an alternative IMP supply from the plastids.The activity of AMPD likely resides in the cytosol, butthe protein has an N-terminal transmembrane do-main and is clearly attached to a membrane (Han et al.,2006).

The following enzymatic reactions for GMP biosyn-thesis probably take place in the cytosol: (1) oxidation ofIMP to xanthosine 59-monophosphate (XMP; Fig. 1) byIMP dehydrogenase (IMPDH; Fig. 3A, no. 3); and (2)amination of XMP to GMP by GMP synthetase (GMPS;Fig. 3A, no. 4). Neither of these enzymes has an ap-parent subcellular targeting peptide, and both were

Figure 1. Structural composition of nucleobases, nucleosides, and nucleotides. For the nucleobases, “R” is simply a proton. Forthe nucleosides, “R” is a sugar moiety that can be ribose or deoxyribose (carrying a proton instead of a hydroxyl group at the 29carbon of the ribose). Nucleotides have up to three phosphate groups esterified to the hydroxyl group of the 59-carbon of thenucleoside sugar determining the prefixmono-, di-, or tri- in the name of themolecule. The terminal phosphate always carries twocharges, irrespective of the number of phosphates present. The pyrimidine nucleobases (upper row) and the purine nucleobases(lower row) are shownwith the groups attached to the heterocycles highlighted in red (oxo groups), blue (amino groups), and gray(methyl group). dTMP, deoxy-TMP; dXMP, deoxy-XMP; dIMP, deoxy-IMP.

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detected in the cytosolic proteome of Arabidopsis (Itoet al., 2011). Also, IMPDH from cowpea nodules wasassociated with the cytosolic fraction (Shelp andAtkins, 1983). GMP is a quite strong competitive in-hibitor of IMPDH (Atkins et al., 1985), maybe resultingin feedback regulation in vivo.

Pyrimidine De Novo Synthesis

The first dedicated reaction of UMP biosynthesis(Fig. 3B) is catalyzed by aspartate transcarbamoylase(ATCase; Fig. 3B, no. 5). The enzyme is located in theplastids. The next enzyme, dihydroorotatase (DHOase;

Fig. 3B, no. 6) was associated to plastids in cell frac-tionation studies (Doremus and Jagendorf, 1985 andreferences on the SUBAweb server; Hooper et al., 2017)but was located in the cytosol when transiently over-expressed in Arabidopsis protoplasts as a GFP-taggedfusion protein (Witz et al., 2012). Maybe DHOase isassociated with the chloroplast membrane via a protein-protein interaction, and the interaction partner is over-whelmed by strong overexpression of DHOase. The nextenzyme in pyrimidine biosynthesis, dihydroorotate de-hydrogenase (DHODH, Fig. 3B, no. 7), is associatedwithmitochondria (Doremus and Jagendorf, 1985;Witz et al.,2012) and likely located on the outer surface of the inner

Figure 2. Schematic overview of plant nucleotide metabolism. Nucleotides are synthesized “de novo” from precursor moleculeslisted in the upper left box. The phosphorylation of nucleoside monophosphates (NMPs) via diphosphates (NDPs) generatesnucleoside triphosphates (NTPs), which serve as building blocks for RNA synthesis and as precursors for the biosynthesis of themetabolites shown in the center (SAM, UDP-Glc, and NADH are given as examples). However, the NTPs, in particular ATP andGTP, are not only precursors for othermetabolites, but are also essential stores of chemical energy in the phosphoanhydride bondsused in a multitude of energetic coupling reactions, as well as important donors of phosphate in kinase reactions (not shown).NDPs can be reduced to dNDPs, which after phosphorylation to dNTPs serve as precursors for DNA biosynthesis. RNA degra-dation in the cytosol releases nucleosidemonophosphates, whereas nucleosides are produced during vacuolar RNAdegradation.Adenosine and adenine are products of biochemical reactions involving SAM. Nonenzymatic decay (depurination) and enzy-matic repair reactions result in nucleoside and nucleobase release from DNA. Nucleobases and nucleosides can be recycled tonucleotides in so-called salvage reactions. Plants are also capable of full nucleotide degradation via certain nucleosides andnucleobases releasing the nitrogen of the nucleobases as ammonia.

Figure 3. Purine and pyrimidine denovo biosynthesis. A, Purine de novobiosynthesis. B, Pyrimidine de novobiosynthesis. Enzymes and transportersinclude brittle1 (BT1; 1), AMP deami-nase (AMPD; 2); IMP dehydrogenase(IMPDH; 3); GMP synthetase (GMPS;4); asparate transcarbamoylase (ATCase;5); dihydroorotatase (DHOase; 6); dihy-droorotate dehydrogenase (DHODH;7);UMP synthase (UMPS; 8); and CTPsynthetase (CTPS; 9). An anchor symboldenotes an association with the respec-tive membrane.

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mitochondrial membrane, as observed for the mam-malian orthologs (Ullrich et al., 2002). UMP synthase(UMPS; Fig. 3B, no. 8), the final enzyme in pyrimidinebiosynthesis, was associated with the plastids and thecytosol, as shown by cell fractionation of pea (Pisumsativum) leaves (Doremus and Jagendorf, 1985) andwas present in the cytosol after overexpression inArabidopsis protoplasts (Witz et al., 2012). Thus, UMPis generated in the cytosol, while it appears that theresponsible enzyme might have some affinity for thechloroplast. Because ATCase (Fig. 3B, no. 5) is feed-back regulated by uridylates, in particular UMP(Doremus and Jagendorf, 1985), the cytosolic uridinenucleotide pool must be tightly connected to theplastidic pool.

CTP biosynthesis requires the phosphorylation ofUMP to UTP (see section on "The Generation of Nu-cleoside Triphosphates"), which is the substrate ofCTP synthetase (CTPS; Fig. 3B, no. 9). There are fiveCTPS isoenzymes in Arabidopsis, and all reside inthe cytosol. For one isoform (CTPS3), the activity andstimulatory allosteric regulation by UTP and GTPhave been recently shown (Daumann et al., 2018).Interestingly, some CTPS isoenzymes form filamen-tous aggregates, called cytoophidia, inactivating theenzyme. In vitro, these are generated in particular inthe presence of CTP, indicating that the enzyme isfeedback regulated by this mechanism (Daumannet al., 2018). Knockout mutants for each CTPS werecharacterized, and except for CTPS2, which showed acomplete block of germination, no phenotypes wereobserved in the single mutants (Daumann et al., 2018),

indicating redundancy of the CTPS enzymes in mostsituations.

The Generation of Nucleoside Triphosphates

The final steps of UMP, GMP, and CTP biosynthesisoccur in the cytosol.With the exception of CTP, which isdirectly synthesized from UTP, purine and pyrimidinenucleoside triphosphate synthesis is achieved byphosphorylation of the respective monophosphates(Fig. 4).

The plastids and the mitochondria, which possesstheir own transcription and translation machineries,must be supplied with ribonucleotides and deoxyribo-nucleotides from the cytosol. Not much is known about(1) the phosphorylation state in which nucleotides aretaken up, (2) which transporters are involved, or (3)whether the concentrations of (desoxy) nucleotidesdiffer in the distinct cellular compartments and howthis may be regulated—subcellular distributions havebeen estimated only for the adenylates (Stitt et al., 1982).Describing the subcellular distribution of the enzymesinvolved in the last two steps of mononucleotidephosphorylation can help in building hypotheses re-garding the exact nucleotide species imported intoorganelles.

The pyrimidine nucleotides, UMP and CMP, arephosphorylated by UMP kinases (UMKs; Fig. 4, Aand B, no. 12) Arabidopsis possesses two evolution-arily distinct families of such enzymes: (1) UMKs re-lated to adenylate kinases (AMKs) encoded by fourgenes; and (2) UMKs related to eubacterial UMP kinases

Figure 4. Synthesis of NTPs anddNTPs. Synthesis of cytidylates (A),uridylates and thymidylates (B), gua-nylates (C), and adenylates (D). RNR(10), ribonucleotide reductase; dNK(11), deoxynucleoside kinase; UMK(12), UMP kinase; NDPK (13), nucle-oside diphosphate kinase; TK (14),thymidine kinase; DHFR-TS (15),dihydrofolate reductase-thymidylatesynthase; TMK (16), thymidylate ki-nase; GMK (17), guanylate kinase;AMK (18), adenylate kinase. The sub-cellular locations where enzymes withthese activities are found are indicated.For TMK, a location in the plastids isonly assumed. The mononucleotides(AMP, GMP, UMP, and CMP) may alsobe derived from salvage reactions (seeFigs. 5 and 6).


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encoded by two genes. The AMK-like UMKs have notyet been characterized, except for a biochemical analysisof UMK3 (At5g26667), which was shown to utilize UMPand CMP as the best substrates (Zhou et al., 1998). Theseenzymes have been predicted to reside in the cytosol andthe mitochondria (Lange et al., 2008). From the eubac-terial UMP kinase family, one member, called plastidUMP kinase (PUMKIN; At3g18680), was shown to belocated in chloroplasts and to have UMK activityin vitro. Interestingly, the enzyme binds certain plastidictranscripts and is involved in plastid RNA metabolism,which may not require its enzymatic function. Mutantsare small and compromised in plastid translation andphotosynthetic performance (Schmid et al., 2019). Theorthologous enzyme in rice is localized in chloroplastsand participates in RNA metabolism, and the corre-sponding loss-of-function mutants are pale green (Zhuet al., 2016; Chen et al., 2018a). Additionally, they containless UDP and more UMP (Dong et al., 2019), suggestingthat UMP phosphorylation in the chloroplast is func-tionally important. The phosphorylation of thymidine59-monophosphate (TMP) is not catalyzed by UMKs butby a dedicated TMP kinase (TMK; Fig. 4B, no. 16). InArabidopsis, a mitochondrial and a cytosolic version aregenerated from a single gene by alternative splicing.Mutation of TMK leads to early seed abortion at thezygote state (Ronceret et al., 2008).The phosphorylation of GMP to GDP and dGMP to

dGDP (Kumar et al., 2000) is catalyzed by guanylatekinases (GMK; Fig. 4C, no. 17), of which plants havetwo different types, a cytosolic type (GKc), and an or-ganelle type dually targeted to plastids and mitochon-dria (GKpm; Fig. 4C). The activity of GKpm in rice, pea,and Arabidopsis is regulated by guanosine 39, 59-bisdiphosphate (ppGpp), a bacterial and plastid sig-naling molecule (Nomura et al., 2014). Suppression ofArabidopsis GKpm (At3g06200) transcripts by RNAinterference results in a pale green or albino phenotype(Sugimoto et al., 2007), emphasizing the importance ofnucleotide monophosphate transport into organelles.Interestingly, the loss-of-function mutant of the riceGKpm gene is pale green but does not exhibit DNAdepletion in the organelles, suggesting that deoxynu-cleotide and ribonucleotide metabolism are not fullylinked (Sugimoto et al., 2007).Adenylate monophosphate is converted by adeny-

late kinase (AMK; Fig. 4D, no. 18) into ADP. Arabi-dopsis has seven AMK isoforms. AMK1, AMK2, andAMK5 are located in the plastids, AMK3 and AMK4reside in the cytosol, and AMK7 is present in the mi-tochondria. For AMK1, a mitochondrial localizationhas also been observed (Carrari et al., 2005; Lange et al.,2008). Because the plastids harbor the de novo synthesisfor AMP, they will not require net import of adenylates.Consistently, the loss-of-functionmutant for AMK2 hasa bleached phenotype (Lange et al., 2008), suggestingthat there is no net ADP or ATP import to compensatefor compromised adenlyate kinase activity in plastids.A strong reduction of plastidic AMK activity in ricealso results in an albino phenotype (Wei et al., 2017).

In potato (Solanum tuberosum), a reduction of plastidicAMK activity led to an increase of the adenylate pool(AMP, ADP, ATP, and ADP-Glc) and increased starchsynthesis in tubers, but it is still not understood howplastidic AMK and adenylate de novo synthesis areconnected (Regierer et al., 2002). Interestingly, it wassuggested that the AMKs might contribute to protectRNA from random misincorporation of methyl-6 Amarks. AMKs are highly selective for AMP versus N6-methyl AMP released during the degradation of RNAspecies carrying this abundant A modification. Theselectivity of the AMKs possibly prevents the formationof N6-methyl ATP, which is a substrate of RNA poly-merase II (Chen et al., 2018b).Recently, a broad-spectrum mononucleotide kinase

only distantly related to the AMKs but with relativelyhigh adenylate kinase activity (At5g60340), was de-scribed. This enzyme was localized in the nucleus, anda knockout mutant was affected in stem elongation(Feng et al., 2012).Plastids and mitochondria possess nucleoside

monophosphate kinases for all nucleotides. Thus, nu-cleoside monophosphates are probably imported intothese organelles and are phosphorylated to dinucleo-tides. Enzymes catalyzing the next step to trinucleo-tides, the nucleoside diphosphate kinases (NDPKs),should therefore be found in plastids and mitochon-dria, as well as in the cytosol. This indeed has beenobserved (Luzarowski et al., 2017). The exact loca-tions of the enzymes have been debated and a detailedphylogenetic analysis suggests the presence of a fourthenzyme type in the endoplasmic reticulum (ER; Dorionand Rivoal, 2015). The NDPKs are multisubstrateenzymes accepting all nucleoside/deoxynucleosidediphosphates (Zrenner et al., 2006), but there is apreference for generating GTP (Kihara et al., 2011),which in the chloroplast may assist in repairing pho-tosystem II (Spetea and Lundin, 2012). Mutation of thegene for the plastidic NDPK in rice results in a palegreen phenotype and a lower photosynthetic rate (Yeet al., 2016; Zhou et al., 2017), but since the chloroplastfunction is partially retained, there also must be nu-cleoside triphosphate import into this organelle. Inter-estingly, NDPKs can also have moonlighting activity asmodulators of gene expression (Dorion and Rivoal,2018).Besides nucleotides, the nucleus and organelles need

deoxynucleotides (dNTPs) for DNA synthesis. dNTPsynthesis requires the reduction of the hydroxyl moietyon the 29 carbon of the ribose by an enzyme complexcalled ribonucleotide reductase (RNR; Fig. 4, no. 10).The RNR complex is comprised of two large regulatory(R1) and two small catalytic (R2) subunits. Mutation ofthe major R2 subunit gene (tso2) results in lower dNTPconcentrations and abnormal plant development, whilethe additional mutation of a further R2 subunit gene(Arabidopsis has three R2 subunit genes in total) is le-thal (Wang and Liu, 2006). The substrates of RNR arethe ribonucleotide diphosphates, suggesting that forCTP, a dedicated phosphatase might exist to support





dCDP synthesis (Fig. 4A). Alternatively, CDP for dCTPsynthesis might be generated from salvage of cytidine(see section on "Purine and Pyrimidine Salvage Me-tabolism"). Interestingly, RNR is subject to a complexallosteric regulation to adjust the correct dNTP poolsizes (Sauge-Merle et al., 1999). In plants, RNR residesexclusively in the cytosol, with the potential to relocateto the nucleus upon exposure to UV radiation (Linckeret al., 2004). The plastidic DNA replication, especially,seems to rely strongly on sufficient RNR activity, be-cause partially compromising the function of the largeRNR subunit by different mutations in the corre-sponding gene resulted in reduced dNTP levels andimpaired chloroplast division in Arabidopsis (Gartonet al., 2007). Consistently, chlorophyll biosynthesis inrice is reduced in mutants of the small RNR subunitgenes (Chen et al., 2015). All dNDPs can be synthesizeddirectly by RNR except for thymidine 59 diphosphate,

because it has no ribonucleotide counterpart. Instead,RNR catalyzes the formation of deoxy-UDP from UDP(Fig. 4B) and deoxy-UMP is methylated at C5 to TMPcatalyzed by thymidilate synthase. In Arabidopsis,three enzymes were recently characterized as thymi-dylate synthases, which are also dihydrofolate reduc-tases (DHFR-TS; Fig. 4B, no. 15), with only two isoformsdisplaying thymidylate synthase activity (Gorelovaet al., 2017). Interestingly, in roots the subcellular lo-cations of all isoforms depends on the developmentalstage of the cells: cytosolic, nuclear, and mitochondrial(but not plastidic) locations have been observed. Thetwo active isoforms seem to be redundant, since only adouble mutant of the respective genes is lethal, whereassingle-gene loss-of-function mutants are phenotypi-cally inconspicuous (Gorelova et al., 2017). Thesubstrate for dihydrofolate reductase-thymidylatesynthase is dUMP (Gorelova et al., 2017), but the

Figure 5. Salvage and degradation ofpurines. A, Reactions of purine nucle-obase and nucleoside salvage, as wellas purine nucleotide degradation, whichoverlaps partially with GMP synthesis.The salvage pathways are highlighted bylight gray shading, and the degradationreactions are encircled in dark gray.Metabolites that can only undergo deg-radation and cannot be salvaged areshown with brown shading. B, Purinering catabolism. The transport steps forurate and (S)-allantoin are not shownexplicitly. APRT (19), adenine phos-phoribosyltransferase; ADK (20), adeno-sine kinase; AMPD (2), AMP deaminase;IMPDH (3), IMP dehydrogenase; GMPS(4), GMP synthetase; HGPRT (21),hypoxanthine guanine phosphoribo-syltransferase; IGK (22), inosine gua-nosine kinase; GMPP (23), GMPphosphatase; GSDA (24), guanosinedeaminase; XMPP (25), XMP phospha-tase; NSH1 (26), nucleoside hydrolase1; NSH2 (27), nucleoside hydrolase2; XDH (28), xanthine dehydrogen-ase; UOX (29), urate oxidase; ALNS(30), allantoin synthase; ALN (31),allantoinase; AAH (32), allantoateamidohydrolase; UGAH (33), ureido-Gly aminohydrolase; UAH (34),ureidoglycolate amidohydrolase.


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RNR provides dUDP. It is unknown which enzymelinks these two processes in vivo. An alternativedUMP source in mitochondria is the deamination ofdCMP, as shown recently in rice (Xu et al., 2014; Niuet al., 2017).

SALVAGE AND DEGRADATION

Metabolic Sources of Nucleosides and Nucleobases

Nucleosides and nucleobases can be released fromnucleotides or nucleic acids during metabolism (Figs. 2,5, and 6) or can be taken up from the environment(Girke et al., 2014), where they can occur in substantialamounts (Phillips et al., 1997).The main metabolic source for most nucleosides is

probably the turnover of RNA, in particular in thevacuole. Vacuolar RNA degradation, for example ofribosomal RNA after ribophagy (Floyd et al., 2015),generates nucleotides that likely are degraded to nu-cleosides by vacuolar phosphatases. The details of thisprocess have not been investigated so far. The tonoplastmembrane possesses a nucleoside exporter (equili-brative nucleoside transporter 1 [ENT1, At1g70330];Bernard et al., 2011) for the release of nucleosides intothe cytoplasm. For adenosine, the turnover ofS-adenosyl Met (SAM), used for methylation reactions,is another important source (Figs. 2 and 5). Aftertransfer of the methyl group from SAM, the resultingS-adenosyl homo-Cys (SAH) is hydrolyzed to homo-Cys and adenosine (Sauter et al., 2013).There are no strong sources for nucleobases in plant

metabolism, except for adenine, which is releasedduring polyamine, nicotianamine, and ethylene bio-synthesis (Sauter et al., 2013). In all three pathwaysSAM is used and 59-methylthioadenosine is generated,which is hydrolyzed to 5-methylthioribose and adenine(Siu et al., 2008). Also the degradation of cytokinins

produces small amounts of adenine (Schmüllinget al., 2003). Purine bases are released from nucleicacids by spontaneous depurination (Barbado et al.,2018), resulting in low amounts of adenine and gua-nine. The metabolic source of hypoxanthine is likely thespontaneous deamination of adenine in DNA, resultingin a hypoxanthine base, which is removed from theDNA by base excision repair (Karran and Lindahl,1980). Similarly, the nonenzymatic deamination of cy-tosine in DNA generates uracil, which is removed bybase excision repair (Fig. 2).

Purine and Pyrimidine Salvage Metabolism

Nucleosides and nucleobases can be converted intonucleotides, which is called “salvage” (Figs. 2, 5, and6). In contrast to de novo biosynthesis of nucleotides,which generates nucleotides from basic metabolites(see above), the salvage reactions recycle nucleobasesand nucleosides derived from metabolism or uptake(see section on "Metabolic Sources of Nucleosidesand Nucleobases") to nucleotides. Nucleobases reactwith activated phosphoribose (PRPP) to the respectivenucleotides—a reaction catalyzed by phosphoribo-syltransferases (PRTs). Adenine PRT (APRT; Fig. 5A,no. 19), hypoxanthine guanine PRT (HGPRT; Fig. 5A,no. 21), and uracil PRT (UPRT; Fig. 6, no. 39) are thethree types of nucleobase-specific enzymes present inplants. Nucleosides are phosphorylated to nucleosidemonophosphates by kinases. Adenosine kinase (ADK;Fig. 5A, no. 20), inosine guanosine kinase (IGK;Fig. 5A, no. 22), and uridine cytidine kinase (UCK;Fig. 6, no. 35) salvage ribonucleotides, whereas thy-midine kinase (TK; Fig. 4B, no. 14) and deoxynucle-oside kinase (dNK; Fig. 4, A, C, and D, no. 11)phosphorylate thymidine and the other three deox-ynucleosides, respectively.

Figure 6. Salvage and degradation of pyrimi-dines. NC-b-Ala, N-carbamyl-b-Ala; malonate-SA, malonate semialdehyde. UCK (35), uridinecytidine kinase; UCPP (36), UMP CMP phospha-tase; CDA (37), cytidine deaminase; NSH1 (26),nucleoside hydrolase 1; PLUTO (38), plastidicnucleobase transporter; UPRT (39), uracil phospho-ribosyltransferase; DPYD (40), dihydropyrimidinedehydrogenase; DPYH (41), dihydropyrimidinehydrolase; b-UP (42), b-ureidopropionase; BAAT(43) b-Ala aminotransferase.





Several salvage enzymes have a critical function forplant metabolism and the mutation of the respectivegenes has severe consequences. Mutation of the genefor the main APRT (Fig. 5, no. 19) activity, APT1(At1g27450), results in male sterility (Gaillard et al.,1998), whereas a strong downregulation increases theresistance to oxidative stress (Sukrong et al., 2012).Deletion or strong downregulation of the gene for themain ADK (Fig. 5, no. 20) enzyme, ADK1 (At3g09820),compromises transmethylation reactions, because theaccumulating adenosine inhibits SAH hydrolase—anenzyme of the SAM cycle. It has been shown that ADK1and SAH hydrolase interact and partially reside in thenucleus, probably mediated by nuclear methyltrans-ferases (Lee et al., 2012). Reduced transmethylationcauses a range of developmental abnormalities (Moffattet al., 2002; Young et al., 2006). Guanine and hypo-xanthine salvage seems to be less critical, becauseHGPRT (Fig. 5, no. 21) mutants are phenotypicallynormal except for a slight delay in germination (Liuet al., 2007; Schroeder et al., 2018). Mutation ofHGPRT leads to guanine but not hypoxanthine accu-mulation in vivo, probably reflecting the fact that gua-nine can only be salvaged, whereas hypoxanthine canalso be degraded (Baccolini and Witte, 2019). IGK hasbeen measured in plant extracts (Fig. 5A, no. 22;Katahira and Ashihara, 2006; Deng and Ashihara,2010), but the corresponding gene is still unknown.Some evidence has been provided that the activity isassociated with the intermembrane space of mito-chondria (Combes et al., 1989).

The only uracil salvage activity in Arabidopsis is lo-cated in plastids and encoded by UPP (UPRT; Fig. 6,no. 39). Mutation of UPP leads to growth arrest in theseedling stage and an albino phenotype (Mainguetet al., 2009). Interestingly, it was recently shown thatthis phenotype is unrelated to the lack of UPRT activityin the mutant, but is caused by the absence of the UPPprotein per se, demonstrating that uracil salvage doesnot play such an essential role for Arabidopsis as pre-viously thought (Ohler et al., 2019). Salvage of uridine ismore prominent than salvage of uracil in Arabidopsis.Uridine and cytidine salvage are performed by dual-specific UCKs (Ohler et al., 2019). These enzymes alsopossess a UPRT-like domain, but they do not haveUPRT activity (Chen and Thelen, 2011). Simultaneousmutation of UCK1 and UCK2 results in dwarf plantsthat fail to reach maturity (Chen and Thelen, 2011).Previously, UCK1 and UCK2 were found to localize inplastids (Chen and Thelen, 2011), but Ohler et al. (2019)demonstrated that these enzymes reside in the cytosol,which was also confirmed in our laboratory (M. Chenand C.-P. Witte, unpublished data).

Interestingly, deoxynucleoside-specific salvage en-zymes also exist (Fig. 4). Thymidine is salvaged bythymidine kinase (TK; Fig. 4B, no. 14) and the mutationof both TK genes is lethal for Arabidopsis (Clausenet al., 2012). However, it is unclear, why thymidinesalvage is of such importance. TK occurs in the cytosol,the mitochondria, and the plastids (Xu et al., 2015) and

is of particular importance for chloroplast mainte-nance when germinating seedlings turn autotrophic(Pedroza-García et al., 2019). The other deoxynucleo-sides are salvaged by an enzyme with broad deoxy-nucleoside specificity (dNK; Fig. 4, no. 11; Clausenet al., 2012), potentially associated with mitochondria(Clausen et al., 2014).

Purine Nucleotide Degradation

Instead of being salvaged, nucleobases and nucleo-sides can also be fully degraded by plants, but forguanine, adenine, and adenosine, a salvage reactionneeds to precede degradation. Adenine and adenosinefirst must be converted to AMP, which can then bedeaminated by AMPD (Fig. 5A, no.2) to IMP as a firststep in degradation. This is necessary because Arabi-dopsis and plants in general lack adenosine deaminase(Dancer et al., 1997; Chen et al., 2018b). Interestingly, forN6-methyl AMP, plants as well as many other eukary-otes possess a special deaminase, calledN6-methyl-AMPdeaminase (MAPDA; Chen et al., 2018b). N6-methylatedadenine is the most frequent modification in mRNA, butit is also present in other RNA species (Chen and Witte,2019). MAPDA is phylogenetically related to adenosinedeaminases and hydrolyzes N6-methyl AMP to IMP, re-moving the aminomethyl group. This example shows thatmodified nucleotides must also have an access route togeneral nucleotide degradation.

From IMP, the purine nucleotide degradation path-way cannot be entered directly in Arabidopsis, butconversion to XMP, and apparently even to GMP, isrequired (Baccolini and Witte, 2019). These recent re-sults show that the route for AMP catabolism and theroute for GMP biosynthesis (partially) overlap. There-fore, branch points of both routes must be controlled,but it is not yet clear how this is achieved. GMPdephoshorylation by a yet unknown phosphatase(GMPP; Fig. 5A, no. 23) initiates purine nucleotide ca-tabolism. At the stage of guanosine, salvage back to thenucleotide level via IGK (Fig. 5A, no. 22) is still possible,but the GMPP and IGK reactions might be spatially ortemporarily separated to avoid a futile cycle. The de-amination of guanosine to xanthosine by guanosinedeaminase (GSDA; Fig. 5A, no. 24; Dahncke and Witte,2013) marks the point of no return, because xanthosinecannot be salvaged and is dedicated for degradation(Yin et al., 2014). Although xanthosine appears to begeneratedmainly byGSDA, there is strong evidence foran alternative route directly from XMP to xanthosinecatalyzed by an XMP phosphatase (XMPP; Fig. 5A,no. 25; Baccolini and Witte, 2019). An XMP-specificphosphatase, which may represent this XMPP, is cur-rently under investigation in our laboratory. In sum-mary, GMP catabolism begins with dephosphorylationand deamination of guanosine, and most AMP isapparently also degraded via GMP, although someAMPmight be dephosphorylated already at the stageof XMP.


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In clear contrast to purine metabolism in many otherorganisms, guanine is not an intermediate of purinenucleotide catabolism in Arabidopsis and probablymost plants. For degradation, guanine must first besalvaged to GMP (Dahncke and Witte, 2013; Baccoliniand Witte, 2019). In addition, in contrast to purinemetabolism in many other organisms, inosine and hy-poxanthine are not major intermediates of purine nu-cleotide catabolism in Arabidopsis, because they arederived not from IMP dephosphorylation (Baccoliniand Witte, 2019), but possibly from transfer RNAturnover and base excision repair of deaminated ade-nine in DNA (Fig. 5A). Because guanine and hypo-xanthine do not play an important role in purinenucleotide degradation, HGPRT (Fig. 5A, no. 21) isdecoupled from purine catabolism in plants (Baccoliniand Witte, 2019), which is in stark contrast to humans,where mutation of HGPRT results in accumulation ofpurine nucleotide breakdown products and severephenotypic consequences, known as the Lesch-NyhanSyndrome (Torres and Puig, 2007).Purine catabolism can lead to the complete disinte-

gration of the purine ring in plants (Fig. 5B; Werner andWitte, 2011) to recycle nitrogen (Soltabayeva et al.,2018), but it is also used to generate the intermediatesuric acid and especially allantoin, which counteractstress by reducing reactive oxygen species (Brychkovaet al., 2008; Watanabe et al., 2014; Irani and Todd, 2016,2018; Lescano et al., 2016; Ma et al., 2016; Casartelliet al., 2019; Nourimand and Todd, 2019). Sometimesalso the accumulation of allantoate has been observed(Alamillo et al., 2010). In tropical legumes like soybean(Glycine max) or common bean (Phaseolus vulgaris), theureides allantoin and allantoate are used as long-distance nitrogen transport compounds for the exportof fixed nitrogen from the nodules (Tegeder, 2014;Carter and Tegeder, 2016) mediated by the ureidepermease (UPS) transporters (Desimone et al., 2002;Schmidt et al., 2006; Collier and Tegeder, 2012). Ureidesalso function in long-distance transport in non-nodulated legumes (Díaz-Leal et al., 2012; Quileset al., 2019) and are probably used in many plantsfor this purpose (Lescano et al., 2016; Redillas et al.,2019).It has recently been shown that xanthosine hydroly-

sis to xanthine and ribose is catalyzed by a cytosolicnucleoside hydrolase heteromer consisting of nucleo-side hydrolase 1 (NSH1; Fig. 5A, no. 26) and NSH2(Fig. 5A, no. 27) in vivo (Baccolini and Witte, 2019).NSH1 has only weak xanthosine and inosine but stronguridine hydrolase activity (Jung et al., 2009; Jung et al.,2011; Riegler et al., 2011; Baccolini and Witte, 2019).However, NSH1 is required to activate NSH2, which isthe stronger xanthosine and inosine hydrolase in thecomplex. Nucleoside catabolism is the major meta-bolic source of ribose, which is recycled to ribose5-phosphate by ribokinase in the plastids (Riggs et al.,2016; Schroeder et al., 2018). Xanthine and hypoxan-thine are catabolized by the same enzyme, xanthinedehydrogenase (XDH; Fig. 5A, no. 28; Urarte et al.,

2015), finally to uric acid in the cytosol. Arabidopsishas a second gene encoding XDH (At4g34900) with noapparent XDH activity in vivo (Hauck et al., 2014). In-terestingly, XDH has been shown to play a dual roleduring powdery mildew pathogen attack on Arabi-dopsis (Ma et al., 2016). In the epidermis, the enzyme ispostulated to operate as an NADH oxidase generatingsuperoxide (Zarepour et al., 2010) to prevent fungalentry, whereas in the mesophyll it works as a XDHproducing urate, which is suggested to function as areactive oxygen species scavenger. It was proposed thatby this mechanism the reactive oxygen species areconfined to the infection site. For further degradation,uric acid must be imported into the peroxisomes, butmolecular details about this import are still unknown.In the peroxisome, urate is oxidized by urate oxidase(UOX; Fig. 5B, no. 29) and is hydrolyzed and decar-boxylated by allantoin synthase (ALNS; Fig. 5B, #30) to(S)-allantoin (Lamberto et al., 2010; Pessoa et al., 2010).Mutation of UOX leads to strong accumulation of uricacid, which is deleterious for peroxisome maintenancein the embryo, leading to a severe suppression of ger-mination and seedling establishment (Hauck et al.,2014)—surprisingly, accumulation of similar amountsof xanthine in Arabidopsis plants lacking XDHdoes notlead to strong phenotypic alterations under standardgrowth conditions (Hauck et al., 2014; Schroeder et al.,2018; Soltabayeva et al., 2018). Allantoin must betransported from the peroxisomes to the ER for furtherdegradation, but it is unclear how this is achieved. Onemay speculate that allantoin accumulation under cer-tain stress conditions might be caused in part by alteredperoxisome-to-ER transport efficiency. However, oneapparent reason for allantoin accumulation is an alteredcatalytic capacity for allantoin generation and degra-dation under stress (Irani and Todd, 2016; Lescanoet al., 2016; Irani and Todd, 2018; Casartelli et al., 2019).In the ER, allantoin is hydrolyzed by four enzymes

(Fig. 5B, nos. 31–34) completely releasing the ringnitrogen as ammonia (Todd and Polacco, 2006; Werneret al., 2008; Serventi et al., 2010; Werner et al., 2010).These enzymes are also responsible for supplying theshoots of tropical legumes with nitrogen exported fromthe nodules as allantoin and allantoate (Werner et al.,2013; Díaz-Leal et al., 2014).

Pyrimidine Nucleotide Degradation

Pyrimidine nucleotide catabolism is initiated byUMP/CMP phosphatase(s) (UCPP; Fig. 6, no.36),which have not yet been identified. Their activity mightbe temporarily and/or spatially separated from UCKs(Fig. 6, no. 35; Ohler et al., 2019) to avoid a futile cycle ofpyrimidine nucleotide dephosphorylation and pyrimi-dine nucleoside salvage. Cytidine is deaminated touridine by a cytosolic cytidine deaminase (CDA; Fig. 6,no. 37). Interestingly, plants can neither degrade norsalvage the free base cytosine (Katahira and Ashihara,2002). Arabidopsis contains several copies of CDA, but





only one copy is functional. The mutation of CDA re-sults in smaller plants, probably because cytidine ac-cumulation is toxic (Chen et al., 2016). Generally, theaccumulation of nucleosides can reduce plant perfor-mance, as has been shown for GSDA (Fig. 5A, no. 24)mutants (Schroeder et al., 2018). Consistently, trans-genic lines with increased vacuolar nucleoside export(Bernard et al., 2011) are smaller than the wild type.

Uridine is hydrolyzed by the cytosolic NSH1 (Fig. 6,no. 26) to uracil and ribose (Jung et al., 2009). NSH2 isnot involved in uridine hydrolysis. NSH1 occurs in twoforms in vivo, either as a homomer (probably a homo-dimer; Kopecná et al., 2013) for uridine hydrolysis, or asa heteromer interacting with NSH2 for xanthosine andinosine hydrolysis (Baccolini and Witte, 2019). Oneshould note that these nucleoside hydrolases usually donot hydrolyze cytidine (Jung et al., 2009) or guanosinein vivo unless these compounds accumulate in cata-bolic mutants (Dahncke and Witte, 2013; Chen et al.,2016; Baccolini and Witte, 2019). Adenosine is a sub-strate of NSH1 and also of 59-methylthioadenosinenucleosidase 2 (MTAN2; Siu et al., 2008) in vitro, butboth enzymes hydrolyze adenosine with very low cat-alytic efficiency. Themain adenosine hydrolytic activityof Arabidopsis resides probably in the apoplast (seebelow).

The plastidic nucleobase transporter (PLUTO; Fig. 6,no. 38) reallocates uracil, probably in symport withprotons from the cytosol, into the plastids for furthermetabolic conversion (Witz et al., 2012). PLUTO be-longs to the Nucleobase:Cation Symporter 1 family andtransports guanine and adenine in addition to uracil,albeit with lower efficiency. There are indications that athiamine precursor, hydroxymethylpyrimidine, is alsoa PLUTO substrate (Beaudoin et al., 2018). Interest-ingly, it was recently reported that PLUTO orthologsfrom two grasses do not transport uracil, but only ad-enine and guanine next to a few other substrates (Rappet al., 2016), indicating that uracil metabolism might beorganized differently in these species. However, oneshould note that definitive evidence for a function ofPLUTO in uracil transport into plastids in vivo has notyet been presented for any plant. Other transporterscapable of uracil transport have been identified, butthey are located in the plasma membrane (Schmidtet al., 2004; Niopek-Witz et al., 2014).

In the plastid, there is a branch point: uracil can eitherbe salvaged by UPRT (Fig. 6, no. 39; see section on"Purine and Pyrimidine Salvage Metabolism") or bedegraded. When uracil is applied from outside, strongcatabolic activity is usually observed (Ashihara et al.,2001; Katahira and Ashihara, 2002). The first reaction,in which the uracil ring is reduced to dihydrouracil bydihydropyrimidine dehydrogenase (DPYD/PYD1;Fig. 5, no. 40) residing in plastids, was shown to be ratelimiting (Tintemann et al., 1985). Compared to mam-malian DPYD, the plant enzyme lacks C-terminal do-mains for cofactor binding, which are involved inelectron delivery to the active site. Therefore, the plantenzyme is probably incomplete and might require a so

far unknown interaction partner for activity. The loss ofactivity when the enzyme is expressed in the cytosolinstead of the plastid is in agreement with this hy-pothesis (Cornelius et al., 2011). Mutants of DPYD/PYD1 show delayed germination and a misregulationof abscisic acid-responsive genes, whereas constitutiveoverexpression results in an increase in growth andseed number (Cornelius et al., 2011). In the next enzy-matic steps, the dihydrouracil ring is opened by dihy-dropyrimidine hydrolase (DPYH/PYD2; Fig. 6, no. 41)and then the carbamino group is hydrolytically re-leased by b-ureidopropionase (b-UP/PYD3; Fig. 6, no.42; Walsh et al., 2001), generating b-Ala. Not onlyuracil, but also 5-methyluracil (thymine), is degraded inthis pathway (Cornelius et al., 2011), resulting inb-aminoisobutyrate instead of b-Ala. The first reaction(DPYD; Fig. 6, no. 40) is located in the plastids, thesecond (DPYH; Fig. 6, no. 41) in the ER, and the third(b-UP; Fig. 6, no. 42) in the cytosol, but it is unclear whysuch a distribution is favorable, and how the metabo-lites are shuttled to these different locations (Zrenneret al., 2009). Recently, the combination of genome-wideassociation data with correlation networks built frommetabolite and transcriptome data identified an ami-notransferase correlatedwith b-Ala. The correspondingmutants accumulated b-Ala, indicating that this mightbe the missing b-Ala aminotransferase (BAAT/PYD4;Fig. 6, no. 43) of pyrimidine catabolism in plants (Wuet al., 2016).

Pyrimidine catabolism is induced by nitrogen star-vation and in senescence (Zrenner et al., 2009; Corneliuset al., 2011), suggesting that, similar to purine nitrogen,pyrimidine nitrogen is also recycled by plants. Whenuracil is given as the sole nitrogen source, its degrada-tion can support the growth of Arabidopsis to a limitedextent (Zrenner et al., 2009).

eATP

Extracellular ATP (eATP) is a signal molecule that iseither actively released upon a stimulus by plant cellsvia exocytosis or transport or is derived from damagedcells (Fig. 7; Cao et al., 2014). A plasma membrane-based nucleotide transporter belonging to the mito-chondrial carrier family (pmANT1; Fig. 7, no. 44) isinvolved in ATP export with physiological relevance, atleast in pollen (Rieder and Neuhaus, 2011). eATP playsa role in stress responses and is perceived by thereceptor-like kinase does not respond to nucleotides1 (DORN1; Fig. 7, no. 50), which recognizes ATP, GTP,and ADP, but not AMP and adenosine (Choi et al.,2014). Interestingly, CTP and NAD are also sensed byplant cells, and a potential receptor for NAD has beenidentified recently (Wang et al., 2017).

The ATP signal might be quenched by an apoplasticapyrase (Riewe et al., 2008a), an enzyme that hydro-lyzes NTPs or NDPs to nNMPs. Seven apyrases areencoded by the Arabidopsis genome (APY1–APY7),and APY1 and APY2 were believed to represent theseextracellular enzymes (Lim et al., 2014). However, by


Witte and Herde



scanning the substrate spectra of the apyrases, onlyAPY3 (Fig. 7, no. 45) showed strong activity with ATP(and other NTPs), though APY5 and APY6 also wereslightly active with NTPs (Chiu et al., 2015). Theseapyrases are therefore possible candidates for theapoplastic enzymes in Arabidopsis. However, othersecreted phosphatases might also be involved, for ex-ample members of the unspecific purple acid phos-phatases (Wang et al., 2011; Del Vecchio et al., 2014).The AMP resulting from ATP dephosphorylation

is hydrolyzed in the apoplast to adenosine by a 59-nucleotidase (Fig. 7, no. 46). An AMP-specific extracel-lular 59-nucleotidase associated to the plasmamembranewas purified from peanut (Arachis hypogaea; Sharmaet al., 1986; Gupta and Sharma, 1996), but the corre-sponding gene has not been identified. Adenosine canbe either taken up via the adenosine proton symporterequilibrative nucleoside transporter 3 (ENT3; Fig. 7,no. 47; Traub et al., 2007; Cornelius et al., 2012) orfurther hydrolyzed by the apoplastic purine-specificNSH3 (Fig. 7, no. 48; Jung et al., 2011) to adenine andribose. NSH3 hydrolyzes inosine more efficiently thanadenosine, whereas a cell wall-bound nucleoside hy-drolase of potato, probably the ortholog of NSH3 inthis plant, was highly specific for adenosine and didnot hydrolyze inosine (Riewe et al., 2008b).Simultaneous genetic blockage of nucleoside uptake

and hydrolysis leads to an accumulation of adenosineand uridine in the apoplast, a reduction of PSII effi-ciency, and a higher susceptibility to the necrotrophicfungus Botrytis cinerea, possibly caused by reduced ex-pression of WRKY33 (Daumann et al., 2015), which isknown to be essential for Botrytis resistance (Liu et al.,2015). Treatment with eATP increases the resistance toBotrytis (Tripathi et al., 2018), and the expression ofWRKY33 and other defense-related genes is reduced in

a DORN1 mutant and boosted in a DORN1 over-expression line upon challenge with eATP (Jewell et al.,2019). Taken together, these observations suggest thatadenosine accumulation in the apoplast dampens theDORN1-mediated response, indicating that ENT3 andNSH3 are required to remove the breakdown productsof eATP signaling. Also the adenine resulting fromadenosine hydrolysis by NSH3 is taken up by plantcells. It is not entirely clear which transporters mediatethis uptake. Possible candidates are azaguanine resis-tant 1 (AZG1) and AZG2 (Fig. 7, no. 49), which havebeen shown to facilitate adenine and guanine uptake inArabidopsis seedlings (Mansfield et al., 2009), ormembers of the nucleobase-ascorbate transporter fam-ily (Niopek-Witz et al., 2014) as well as of the purinepermease (PUP) family (Girke et al., 2014). Interest-ingly, fungi seem to be able to influence the purinergicsignaling in the apoplast by interfering with apoplasticnucleotide metabolism via the excretion of nucleotida-ses to improve colonization (Nizam et al., 2019).

CONNECTIONS TO CYTOKININ HOMEOSTASIS

The biosynthesis of the cytokinins involves the gen-eration of N6-modified AMP, carrying an isoprenoidgroup (Sakakibara, 2005). However, cytokinin ribotidesor ribosides are inactive—the free modified base is theactive hormone binding to the receptors (Yamada et al.,2001; Romanov et al., 2018). The question ariseswhether the enzymes that are employed for cytokininhomeostasis (activation from ribotides and inactivationto ribosides/ribotides) are the same as for the metabo-lism of adenine nucleotides.It was shown that a cytokinin ribotide-specific en-

zyme (cytokinin riboside 59-monophosphate phos-phoribohydrolase, called lonely guy [LOG]) can releasethe active cytokinin from the ribotide (Kurakawa et al.,2007; Kuroha et al., 2009). A recent report demonstratedthat mutation of the seven genes coding for functionalLOGs in Arabidopsis resulted in a phenotype thatcannot be attenuated by exogenous cytokinin ribotides,suggesting that the hydrolysis by LOGs is the mainpathway of cytokinin activation (Osugi et al., 2017).Therefore, the cytosolic nucleoside hydrolases do notseem to be involved in cytokinin activation, although itcould be shown that cytokinin ribosides are substratesin vitro, catalyzed with comparatively low efficiency(Jung et al., 2009; Kopecná et al., 2013). Consistently,cytokinin-related phenotypes were not observed innucleoside hydrolase mutants (Riegler et al., 2011).However, long-distance transport of cytokinins mayinvolve an activation of cytokinin ribosides/ribotidesin the apoplast prior to uptake or perception (Romanovet al., 2018). In Arabidopsis, NSH3, located in the apo-plast (see section titled “eATP”), has been shown tohydrolyze adenosine, but cytokinin ribosides have notbeen assessed (Jung et al., 2011). Interestingly, an apo-plastic nucleoside phosphorylase was isolated frompotato that converted cytokinin ribosides to cytokinins

Figure 7. Excretion, perception, and degradation of extracellular ATP.pmANT1 (44), plasmamembrane adenine nucleotide transporter; APY3(45), apyrase 3; 59 NT (46), 59-nucleotidase; ENT3 (47), equilibrativenucleoside transporter 3; NSH3 (48), nucleoside hydrolase 3, AZG1/2(49), azaguanine resistant 1 / azaguanine resistant 2, DORN1 (50), doesnot respond to nucleotides 1.





and ribose-1-phosphate in the presence of phosphate,and that can also work in the synthesis direction of ri-bosides (Bromley et al., 2014). The enzyme preferredcytokinins/cytokinin ribosides over adenine/adenosineas substrates and is supposedly involved in cytokinin-mediated tuber endodormancy. Close homologs inArabidopsis (e.g. At4g28940) are predicted to be lo-cated in the apoplast (SUBA web server; Hooper et al.,2017), but have not yet been characterized.

The inactivation of cytokinins is inter alia achieved bytransferring a phosphoribosyl moiety onto the activecytokinin, resulting in an inactive cytokinin-ribotide.This reaction is at least partially performed by an APRT(Fig. 5A, no. 19), because (1) APT1 mutants convertcytokinins less efficiently to the respective ribotides(Moffatt et al., 1991), (2) APT1 loss-of-function plantscontain more active cytokinins (Zhang et al., 2013), and(3) APT1 mutants have cytokinin-related phenotypes(Gaillard et al., 1998a). APT1 is also clearly involved inthe salvage of adenine, which is less efficiently con-verted to AMP in an APT1mutant (Moffatt et al., 1991)and is slightly more abundant in a mutant plant withreduced APT1 activity (Sukrong et al., 2012). In con-clusion, APT1 participates in adenine and cytokininmetabolism and the question arises how one enzymecan serve both distinct metabolic roles adequately.

There is also some evidence that ADK (Fig. 5A, no. 20)contributes to cytokinin homeostasis, because plantswith reduced ADK activity show cytokinin-relatedphenotypes and contain more cytokinin ribosides(Schoor et al., 2011). However, ADK is also involved inthe maintenance of transmethylation activity; there-fore, an indirect impact of reduced ADK activity oncytokinin homeostasis cannot be fully excluded.

CONCLUSIONS

The synthesis, interconversion, and degradation ofnucleotides is intrinsically linked with the propagationand reading of genetic information, with energy me-tabolism including the metabolic activation of manybiomolecules, but also with methylation reactions, sig-nal transduction, the recycling of nitrogen, and themodification of oxidative stress.We are getting closer tocompleting a full inventory of enzymes involved inplant nucleotide metabolism, but we are far from un-derstanding how these enzymes operate together toachieve nucleotide homeostasis. There is only frag-mentary information about regulation on all levels,transport processes are incompletely defined, and theorganization of nucleotide metabolism at the tissue andorgan levels is not well understood (see OutstandingQuestions). These issues need to be addressed to allowa better integration of nucleotide metabolism into amolecular model of plant physiology.

ACKNOWLEDGMENTS

The authors thank Henryk Straube for critical reading of the manuscript.

Received August 1, 2019; accepted October 15, 2019; published October 22,2019.

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