modulation of the poly(adp-ribosyl)ation reaction via the

Modulation of the Poly(ADP-ribosyl)ation Reactionvia the Arabidopsis ADP-Ribose/NADHPyrophosphohydrolase, AtNUDX7, Is Involvedin the Response to Oxidative Stress1[OA]

Kazuya Ishikawa, Takahisa Ogawa, Eisuke Hirosue, Yasumune Nakayama, Kazuo Harada,Eiichiro Fukusaki, Kazuya Yoshimura, and Shigeru Shigeoka*

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nara 631–8505, Japan (K.I.,T.O., S.S.); Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka565–0871, Japan (E.H., Y.N., K.H., E.F.); and Department of Food and Nutritional Science, College ofBioscience and Biotechnology, Chubu University, Kasugai, Aichi 487–8501, Japan (K.Y.)

Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADP-ribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed(Pro35S:AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused byparaquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro35S:AtNUDX7 plants but increased in the KO-nudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro35S:AtNUDX7 and KO-nudx7 plants showedincreased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib)in the Pro35S:AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants.Depletion of NAD+ and ATP resulting from the activation of the PAR reaction under oxidative stress was completelysuppressed in the Pro35S:AtNUDX7 plants. Accumulation of NAD+ and ATP was observed in the KO-nudx7- and3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors,AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under bothnormal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3,AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nucleardivisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD+ by NADHturnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD+ levels by supplying ATP vianucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damagevia modulation of the PAR reaction.

Reactive oxygen species (ROS) are by-products ofnormal metabolic processes, including chloroplastic,mitochondrial, and plasma membrane-linked elec-tron transport systems, in all aerobic organisms(Gutteridge and Halliwell, 1989). Although the pro-

duction and destruction of ROS are in balance, theimposition of biotic and abiotic stressful conditionscan give rise to excess concentrations of ROS, leadingto an imbalance of production and scavenging mech-anisms (Mittler, 2002; Mullineaux and Karpinski, 2002;Kroj et al., 2003; Mahalingam et al., 2003). Excess ROS,leading to oxidative stress, can damage organelles,oxidize proteins, nick DNA (single-base DNA dam-age), deplete antioxidant levels, and ultimately triggercell death (Gutteridge and Halliwell, 1989). Recently,ROS have been recognized as important signalingmolecules that control diverse signaling pathwaysinvolved in a variety of cellular responses such asprogrammed cell death, pathogen defense, and hor-mone signaling (Foyer and Noctor, 2005; Kwak et al.,2006; Torres et al., 2006). In addition, oxidative stresscauses dramatic inhibition of the tricarboxylic acidcycle and large sectors of amino acid metabolismfollowed by backing up of glycolysis and diversionof carbon into the oxidative pentose phosphate path-

1 This work was supported by a Grant-in-Aid for ScientificResearch on Priority Areas (grant no. 19039032 to S.S.) from theMinistry of Education , Culture, Sports, Science, and Technology ofJapan, by Research Fellowships from the Japan Society for thePromotion of Science for Young Scientists (grant no. 18–1015 to T.O.),and by Core Research for Evolutional Science and Technology, JapanScience and Technology Corporation (to S.S.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Shigeru Shigeoka ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.140442

Plant Physiology�, October 2009, Vol. 151, pp. 741–754, www.plantphysiol.org � 2009 American Society of Plant Biologists 741 www.plantphysiol.orgon April 11, 2018 - Published by Downloaded from

Copyright © 2009 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

way (Baxter et al., 2007). Therefore, organisms havedeveloped efficient systems to keep ROS levels incheck and repair damage from attack by ROS.

Among various defense systems against attackby ROS, the poly(ADP-ribosyl)ation (PAR) of pro-teins by poly(ADP-Rib)polymerase (PARP), by whichbranched polymers of ADP-Rib are attached usingb-NAD+ to a specific amino acid residue of an acceptorprotein, is a posttranslational modification for re-sponding early to DNA damage, such as single-strandDNA break and resealing, caused by oxidative stressand, thus, is crucial for genomic integrity and cellsurvival (Qin et al., 2008). PARP detects DNA strandbreaks and converts the damage into intracellularsignals that can activate DNA repair programs or celldeath, according to the severity of the injury, via thePAR reaction of nuclear proteins involved in chroma-tin architecture and DNA metabolism and interactswith the x-ray repair cross complementing factor 1(XRCC1), an adaptor protein that also has two inter-faces with two important single-strand DNA break(SSB) repair (SSBR)/base excision repair (BER) en-zymes: DNA ligase and DNA polymerase b (Caldecottet al., 1995, 1996; Kubota et al., 1996; Masson et al.,1998). DNA polymerase b fills the single nucleotidegap, preparing the strand for ligation by a complex ofDNA ligase III and XRCC1 (Winters et al., 1999;Thompson and West, 2000). Thereby, the fast recruit-ment of SSBR/BER factors is archived in the site ofthe lesion. Modifications of proteins with poly(ADP-Rib) are reversed by poly(ADP-Rib) glycohydrolase(PARG), by which ADP-Rib polymers are hydrolyzedto free ADP-Rib, since incorrect signal transduction iscaused by excessive accumulation of poly(ADP-Rib)modification (Davidovic et al., 2001). However, it hasbeen reported that a massive PAR reaction results inthe overconsumption of NAD+ and ATP and, ulti-mately, in energy depletion causing necrotic cell death(Ha and Snyder, 1999; Virag and Szabo, 2002; De Blocket al., 2005).

Nudix (for nucleoside diphosphates linked to somemoiety X) hydrolases catalyze the hydrolysis of intactand oxidatively damaged nucleoside diphosphatesand triphosphates, nucleotide sugars, coenzymes, di-nucleoside polyphosphates, and RNA caps in variousorganisms such as bacteria, yeast, algae, nematodes,vertebrates, and plants (Bessman et al., 1996; Xu et al.,2004; Kraszewska, 2008). We have previously reportedthe characteristics of cytosolic Nudix hydrolases(AtNUDX1–AtNUDX11) in Arabidopsis (Arabidopsisthaliana; Ogawa et al., 2005). Among them, the recom-binant AtNUDX7 showed high affinity for ADP-Riband NADH as substrates in vitro, converting NADHto a reduced form of nicotinamide mononucleotide(NMNH) plus AMP andADP-Rib to AMP plus Rib 5-P(Ogawa et al., 2005). AtNUDX7 was expressed morestrongly in leaf than in stem and root. Therefore, theenzyme might be involved in nucleotide recyclingrelating to the metabolism of NADH and/or poly(ADP-Rib).

Recent studies revealed that the actions ofAtNUDX7 (At4g12720) are closely related to immuneresponses to pathogens. Knockout of AtNUDX7(KO-nudx7) in Arabidopsis plants led to deleteriousinference for cells, such as microscopic cell death,constitutive expression of pathogenesis-related genes,resistance to bacterial pathogens, and accumulation ofNADH (Jambunathan and Mahalingam, 2006). Fur-thermore, AtNUDX7 exerted a negative regulatoryeffect on EDS1 signaling, which controls the activationof defenses and programmed cell death conditionedby intracellular Toll-related immune receptors thatrecognized specific pathogen effectors (Bartsch et al.,2006). More recently, Ge et al. (2007) reported thatKO-nudx7 plants show heightened defense responses,which are both dependent on and independent of theaccumulation of NPR1 and salicylic acid, to patho-genic attack. On the other hand, Adams-Phillips et al.(2008) reported that KO-nudx7 plants exhibit a reducedhypersensitive-response phenotype, although thegrowth of both virulent and avirulent pathogens issuppressed in the plants. These findings support thehypothesis that regulation of the metabolism ofNADH and/or ADP-Rib by Nudix hydrolases is im-portant for stress-related defense systems in higherplants. However, the direct actions of the enzymes onstress responses are not established yet.

In this study, to assess the functions of ArabidopsisNudix hydrolases having ADP-Rib and NADH pyro-phosphohydrolase activities under normal conditionsand oxidative stress, we analyzed the effect of theoverexpression or disruption of AtNUDX7 on levels ofADP-Rib, NAD(H), and ATP as well as PAR activityand oxidative stress tolerance in Arabidopsis. The ev-idence presented here suggests that AtNUDX7 servesto balance between NADH and NAD+ by NADH turn-over under normal conditions. In addition, AtNUDX7functions in the maintenance of NAD+ levels by sup-plying ATP via nucleotide recycling from free ADP-Rib molecules and the modulation of the PAR reaction,thereby regulating the DNA repair pathways, in re-sponse to oxidative stress.

RESULTS

Characteristics of AtNUDX7-Overexpressedor -Disrupted Arabidopsis Plants

We have previously demonstrated that theAtNUDX7mRNA is expressed ubiquitously in all planttissues (Ogawa et al., 2005). As shown in Figure 1B, themRNA and protein (31.8 kD) of AtNUDX7 in theleaves of the wild-type Arabidopsis plants were de-tected by semiquantitative reverse transcription (RT)-PCR and western-blot analysis, respectively. Thepyrophosphohydrolase activities toward ADP-Rib andNADH in the crude extracts prepared from Arabidop-sis leaves were 63.2 6 1.7 and 95.6 6 3.0 nmol min21

mg21 protein, respectively (Fig. 1D), consistent with theresults reported by Ogawa et al. (2009).

Ishikawa et al.

742 Plant Physiol. Vol. 151, 2009 www.plantphysiol.orgon April 11, 2018 - Published by Downloaded from



In order to clarify the functions of AtNUDX7, aT-DNAmutant ofAtNUDX7 (SALK_046441;KO-nudx7)was obtained from the SIGnAL project (signal.salk.edu/tabout.html). The mutant contained a T-DNAinsert in the first exon of AtNUDX7 (Fig. 1A). Theinsertion resulted in a complete loss of AtNUDX7expression in the KO-nudx7 plants (Fig. 1B). The py-rophosphohydrolase activities toward ADP-Rib andNADH in the KO-nudx7 plants were reduced signifi-cantly to approximately 76.9% and 46.9%, respectively,of the levels in wild-type plants (Fig. 1D). These resultssuggest that AtNUDX7 is the predominant ADP-Rib/NADH pyrophosphohydrolase under normal condi-tions in Arabidopsis cells. No difference was observedin growth or morphology between the control plantsand these knockout plants throughout the cultivationperiod under normal conditions (data not shown).Next, we generated transgenic Arabidopsis plants

overexpressing AtNUDX7 under the control of the

cauliflower mosaic virus 35S promoter (Pro35S:AtNUDX7). Northern-blot analysis showed that thelevels of the AtNUDX7 transcript in the T3 generationof Pro35S:AtNUDX7-5-1, -5-2, and -7-1 plants are ap-proximately 3.7-, 5.6-, and 1.7-fold higher, respectively,than levels in the control plants (transformed with theempty vector; Fig. 1C). By western-blot analysis, alarge amount of the AtNUDX7 protein was detectedin the extracts prepared from the leaves of Pro35S:AtNUDX7-5-1, -5-2, and -7-1 plants (Fig. 1C). Thelevels of protein in the transgenic plants were wellcorrelated with the levels of the transcript. The pyro-phosphohydrolase activities toward ADP-Rib andNADH in the Pro35S:AtNUDX7 plants were approxi-mately 1.2- to 2.5-fold and 1.2- to 2.0-fold, respectively,higher than levels in the control plants (Fig. 1D). Nodifference was observed in growth or morphologybetween the control and these transgenic plants undernormal conditions (data not shown).

Figure 1. Characteristics of AtNUDX7-overexpressed or -disrupted Arabidopsis plants. A, T-DNA insertion site in theArabidopsis KO-nudx7 plants. Exons and introns are represented by boxes and lines, respectively. The deduced start (AUG)and stop (UGA) codons and each nucleotide number in the mRNA are indicated. B, Semiquantitative RT-PCR (top) and western-blot (bottom) analyses of the AtNUDX7 mRNA in wild-type (WT) and KO-nudx7 plants. The wild-type and KO-nudx7 plantswere grown on MS medium for 2 weeks under long-day conditions and then used for the analysis. Semiquantitative RT-PCRanalysis was performed using specific primers for AtNUDX7 and Actin2 in a reaction involving 27 cycles of 95�C for 60 s, 55�Cfor 60 s, and 72�C for 60 s, followed by 72�C for 10 min. Equal loading of each amplified cDNAwas determined with the controlActin2 PCR product. The AtNUDX7 protein was detected by western blotting using a specific polyclonal antibody raised againstthe recombinant AtNUDX7 protein. C, Northern-blot (top) and western-blot (bottom) analyses of the AtNUDX7 protein incontrol (transformed with the empty vector) and Pro35S:AtNUDX7 plants. The control and Pro35S:AtNUDX7 (T3 generations ofindependent transformed lines Pro35S:AtNUDX7-5-1, -5-2, and -7-1) plants were grown on MS medium for 2 weeks under long-day conditions and then used for the analysis. The northern-blot analysis was carried out using the full-length AtNUDX7 cDNAfragment as a probe. Equal loading of each RNA was determined with the Actin2 mRNA. D, ADP-Rib and NADHpyrophosphohydrolase activities in the leaves of wild-type, control, Pro35S:AtNUDX7, and KO-nudx7 plants. Data are means6SD for three individual experiments (n = 3). Different letters indicate significant differences (P , 0.05).

Modulation of Poly(ADP-ribosyl)ation Reaction via AtNUDX7

Plant Physiol. Vol. 151, 2009 743 www.plantphysiol.orgon April 11, 2018 - Published by Downloaded from



Physiological Substrates for AtNUDX7

We have demonstrated that the recombinantAtNUDX7 protein hydrolyzes both ADP-Rib andNADH in vitro (Ogawa et al., 2005). To clarify thephysiological substrate for AtNUDX7 in situ, we an-alyzed the intercellular levels of ADP-Rib and NADHin Pro35S:AtNUDX7 and KO-nudx7 plants under nor-mal conditions. NADH accumulated to higher levelsin the KO-nudx7 plants than in the control plants (Fig.2A). In contrast, the amounts of NADH in the Pro35S:AtNUDX7 plants were remarkably decreased com-pared with those in the control plants. Accordingto capillary electrophoresis-electrospray-tandem massspectrometry (CE-ESI-MS/MS) analysis, the levelsof free ADP-Rib in the KO-nudx7 and the Pro35S:AtNUDX7 plants were significantly higher and lower,respectively, than those in the control plants (Fig. 2B).

Expression of AtNUDX7 in Response to Various Typesof Stress

To assess the involvement of AtNUDX7 in defensesystems against various types of stress, we analyzedthe changes in the expression levels of AtNUDX7 byquantitative RT-PCR under various stressful condi-tions (Fig. 3A). We have verified that expression ofPARP and PARG is induced under the stressful con-ditions analyzed here (Ogawa et al., 2009). The ex-pression of AtNUDX7 was induced rapidly underconditions of high-light illumination (1,600 mE m22

s 21), drought (dehydration on a paper towel), salinity(250 mM NaCl), and by treatment with 3 mM paraquat(PQ; an agent producing O2

2) under high-light illu-

mination, suggesting the participation of excess ROSin the expression. The manipulation for transferringplants in the stress treatments had no effect on theAtNUDX7 expression (data not shown). Western-blotanalysis showed that the levels of AtNUDX7 proteinwere increased by the treatment with PQ (Fig. 3B).Similarly, the pyrophosphohydrolase activities towardboth ADP-Rib and NADH in the plant extracts in-creased by treatment with PQ (Fig. 3C).

Effect of Overexpression or Knockout of AtNUDX7 onOxidative Stress Tolerance

Next, we evaluated the contribution of AtNUDX7 tooxidative stress tolerance using Arabidopsis plants inwhich AtNUDX7 was overexpressed or disrupted. Inorder to show significant differences in stress tolerancebetween transgenic and control Arabidopsis plants,the oxidative stress by treatment with 3 mM PQ undernormal light (100 mEm22 s 21) was performed in Pro35S:AtNUDX7 plants, because the stress by treatmentwith 3 mM PQ under high light (1,600 mE m22 s 21)for the analysis of AtNUDX7 expression caused read-ily severe oxidative damage to plants. As assessed byphenotype, chlorophyll content, and survival rate, thePro35S:AtNUDX7-5-1 and -5-2 plants having high ex-pression levels of AtNUDX7 clearly showed enhancedtolerance to the oxidative stress comparedwith the con-trol plants (Fig. 4, A–C). Since the 3-aminobenzamide(3-AB)-treated and KO-nudx7 plants were more sensi-tive to oxidative stress than the control plants, stressfulconditions by treatment with 2 mM PQ under normallight (100 mE m22 s 21) were used for the evaluation ofoxidative stress tolerance. In contrast to the Pro35S:AtNUDX7 plants, the KO-nudx7 plants showed en-hanced sensitivity to the stress, while no destruction ofchlorophyll was observed in the leaves of wild-typeplants after exposure to the stress (Fig. 4, D–F).

Changes in the Intracellular Levels of NADH, NAD+,ADP-Rib, Poly(ADP-Rib), ATP, and Antioxidants in

the Arabidopsis Plants in Which AtNUDX7 WasOverexpressed or Disrupted under Oxidative Stress

Levels of NADH in the KO-nudx7 and Pro35S:AtNUDX7 plants were significantly high and low,respectively, under stressful conditions (Fig. 2A). Thelevels of free ADP-Rib in the Pro35S:AtNUDX7 plantsweremarkedly low comparedwith those in the controlplants under stressful conditions (Fig. 2B). The levelsof free ADP-Rib in the KO-nudx7 plants were signifi-cantly high under stressful conditions.

Next, we analyzed the amount of poly(ADP-Rib),reflecting the degree of the PAR reaction, in the Pro35S:AtNUDX7 and KO-nudx7 plants under oxidative stresscaused by the treatment with 3 mM PQ. The amount ofpoly(ADP-Rib) in the control plants was increasedunder stressful conditions. The amount in the Pro35S:AtNUDX7 and KO-nudx7 plants was considerablylarger and smaller, respectively, than that in the control

Figure 2. The levels of intracellular NADH and ADP-Rib under normalconditions and oxidative stress. Two-week-old Arabidopsis plants weregrown on MS medium containing 3 mM PQ for 7 d under long-dayconditions. The NADH (A) and ADP-Rib (B) levels in the control, KO-nudx7, and Pro35S:AtNUDX7 plants were determined as described in“Materials and Methods.” Data are means 6 SD for at least threeindividual experiments (n = 3–9). Different letters indicate significantdifferences (P , 0.05). FW, Fresh weight.

Ishikawa et al.




plants under normal and stressful conditions (Fig. 5A),indicating a positive correlation between the expres-sion levels of AtNUDX7 and the levels of the PARreaction.Since the activation of PARP results in the overcon-

sumption of NAD+ and ATP (Ha and Snyder, 1999;Virag and Szabo, 2002; De Block et al., 2005), weanalyzed the levels of NAD+ and ATP in the Pro35S:AtNUDX7 and KO-nudx7 plants under oxidativestress. The levels of both NAD+ and ATP in the controlplants gradually decreased under stressful conditions(Fig. 5, B and C). In contrast, the decrease of NAD+wascompletely suppressed in the Pro35S:AtNUDX7 plants.Interestingly, NAD+ accumulated to higher levels inthe KO-nudx7 plants than in the control plants undernormal and stressful conditions. The decrease in thelevels of ATP under stressful conditions was com-pletely suppressed in the Pro35S:AtNUDX7 plants, al-though the level of ATP was significantly lower thanthat in the control plants even under normal condi-tions (Fig. 5C). The level of ATP in the KO-nudx7 plantswas similar to that in the control plants under normalconditions and slightly decreased under stressful con-ditions.The redox homeostasis including antioxidative sta-

tus in the plant cells is enormously affected by theintracellular NADH level. To obtain more informationon the relationship between the cellular redox stateand the actions of ADP-Rib/NADH pyrophosphohy-drolases, we determined levels of ascorbate (AsA) andglutathione (GSH), as important antioxidants, in the

plants in which AtNUDX7 was overexpressed ordisrupted. However, the levels of AsA, oxidized AsA(dehydroascorbate [DAsA]), GSH, and oxidized GSH(GSSG) in the Pro35S:AtNUDX7 and KO-nudx7 plantswere unaltered compared with those in the controlplants under both normal conditions and oxidativestress caused by the treatment with 3 mM PQ (100 mEm22 s 21; data not shown).

Effect of Overexpression or Disruption of AtNUDX7 onGene Expression via Modulation of the PAR Reaction

Since activation of the PAR reaction is involved inthe repair of DNA damaged by oxidative stress, weanalyzed the effect of the overexpression or depletionof AtNUDX7 on the expression of the genes encod-ing factors involved in the repair: the x-ray repaircross-complementing factors (AtXRCC1 [At1g80420],AtXRCC2 [At5g64520], and AtXRCC3 [At5g57450]),the Escherichia coli RecA homologs (AtRAD51[At5g20850], AtRAD51B [At2g28560], AtRAD51C[At2g45280], AtRAD51D [At1g07745], and AtDMC1[At3g22880]), and the yeast MND1 homolog (AtMND1[At4g29170]; Fig. 6). The expression levels of AtXRCC1and -2 were significantly increased and decreased inthe Pro35S:AtNUDX7 and KO-nudx7 plants, respec-tively, compared with those in the control plants un-der both normal conditions and oxidative stress. Onthe other hand, the levels of AtXRCC3, AtRAD51,AtDMC1, and AtMND1 in the Pro35S:AtNUDX7 andKO-nudx7 plants were lower and higher, respectively,

Figure 3. Changes in the expression levels of AtNUDX7 under various types of stress. A, Quantitative RT-PCR analysis ofAtNUDX7 expression in response to oxidative stress. Two-week-old Arabidopsis plants were subjected to various forms of stress:treatment with PQ (3 mM under illumination at 1,600 mE m22 s 21), salinity (250 mM NaCl), high-light intensity (1,600 mE m22

s 21), and drought (dehydration on a paper towel). Total RNA extracted from Arabidopsis leaves was converted into first-strandcDNA using the oligo(dT)20 primer. A quantitative RT-PCR analysis was carried out to determine the expression level ofAtNUDX7. The relative amounts were normalized to Actin2 mRNA. Data are means 6 SD for three individual experiments (n =3). B, Western-blot analysis of the AtNUDX7 protein in the leaves of Arabidopsis plants. Two-week-old Arabidopsis plants weregrown on MS medium containing 3 mM PQ under illumination at 1,600 mE m22 s 21 for 3 h. C, ADP-Rib and NADHpyrophosphohydrolase activities in the leaves of Arabidopsis plants under the PQ treatment. Data are means 6 SD for threeindividual experiments (n = 3). Different letters indicate significant differences (P , 0.05).





than those in the control plants under normal condi-

tions or oxidative stress. There was no difference inthe levels of AtRAD51B, AtRAD51C, and AtRAD51D

between the controls and transformants (data notshown).

Effect of Inhibition of PARP on Oxidative

Stress Tolerance

Next, we evaluated the effect of suppression ofthe PAR reaction on oxidative stress tolerance ofArabidopsis plants. It has been reported that 3-AB,

Figure 4. Effects of overexpression or disruption of AtNUDX7 on oxidative stress tolerance. A, Phenotypes of the control andPro35S:AtNUDX7 plants after oxidative stress caused by PQ treatment. Seven-day-old seedlings were grown on MS mediumcontaining 3 mM PQ for 7 d under long-day conditions. The seedlings were grown then on MS medium without PQ for anadditional 7 d. B, Survival rates of the control and Pro35S:AtNUDX7 plants under the PQ treatment. C, Chlorophyll contents of thecontrol and Pro35S:AtNUDX7 plants under normal conditions and oxidative stress. Data are means 6 SD for three individualexperiments (n = 3). Different letters indicate significant differences (P , 0.05). D, Phenotypes of the wild-type (WT) and KO-nudx7 plants after oxidative stress caused by PQ treatment. Two-week-old Arabidopsis plants were grown on MS mediumcontaining 2 mM PQ for 7 d under long-day conditions. The plants were grown then on MS medium without PQ for an additional7 d. E, Survival rates of wild-type and KO-nudx7 plants under PQ treatment. F, Chlorophyll contents of wild-type and KO-nudx7plants under normal conditions and oxidative stress. Data are means 6 SD for three individual experiments (n = 3). Differentletters indicate significant differences (P , 0.05). FW, Fresh weight.

Ishikawa et al.




a commercially available inhibitor, is used success-fully to suppress the PARP activity in plants (Jagtapand Szabo, 2005). Phenotype, chlorophyll content,and survival rate of Arabidopsis plants showed thattreatment with 3-AB enhanced the sensitivity to ox-idative stress caused by PQ treatment under normallight conditions (Fig. 7). The amount of poly(ADP-Rib) in the wild-type Arabidopsis plants treated with3-AB was significantly smaller than that in the un-treated wild-type plants under normal conditionsand oxidative stress (Fig. 8A). Furthermore, NAD+

accumulated to higher levels in the 3-AB-treatedplants than in the untreated plants under normaland stressful conditions, although the NADH levelsin the 3-AB-treated plants were similar to those in the

untreated plants (Fig. 8, B and C). The levels of ATP inthe 3-AB-treated plants were similar to those in theuntreated plants under normal conditions andslightly decreased under stressful conditions (Fig.8D). Notably, the 3-AB-treated plants showed en-hanced sensitivity to oxidative stress compared withthe untreated plants (Fig. 7). The expression levels ofAtXRCC1 and -2 were significantly decreased in the3-AB-treated plants compared with those in the un-treated plants under both normal conditions and ox-idative stress (Fig. 9). On the other hand, the levels ofAtXRCC3, AtRAD51, AtDMC1, and AtMND1 in the3-AB-treated plants were higher than those in theuntreated plants under normal conditions or oxidativestress.

Figure 5. The levels of poly(ADP-Rib),NAD+, and ATP under normal conditionsand oxidative stress. Experimental condi-tions are the same as in Figure 2. The levelsof poly(ADP-Rib) (A), NAD+ (B), and ATP(C) in the control, KO-nudx7, and Pro35S:AtNUDX7 plants were determined as de-scribed in “Materials and Methods.” Dataare means6 SD for at least three individualexperiments (n = 3–6). Different lettersindicate significant differences (P , 0.05).FW, Fresh weight.

Figure 6. Changes in the expression ofgenes related to DNA repair systemsvia modulation of the PAR reaction byAtNUDX7. Experimental conditions arethe same as in Figure 2. Quantitative RT-PCR analysis was carried out to determinethe expression levels of genes encodingthe factors involved in the repair of DNA:AtXRCC1, AtXRCC2, AtXRCC3, AtRAD51,AtDMC1, and AtMND1 in the control,KO-nudx7, and Pro35S:AtNUDX7 plants.Relative amounts were normalized toActin2 mRNA. Data are means 6 SD forthree individual experiments (n = 3). Dif-ferent letters indicate significant differ-ences (P , 0.05).





DISCUSSION

AtNUDX7 Functions as an ADP-Rib/NADHPyrophosphohydrolase in Vivo

First, to clarify the physiological substrate for acytosolic Nudix hydrolase, AtNUDX7, in situ, we

analyzed the intercellular levels of NADH and ADP-Rib in Pro35S:AtNUDX7 and KO-nudx7 plants undernormal conditions and oxidative stress (Fig. 2). Ourfindings strongly suggest that AtNUDX7 functions inthe hydrolysis of both ADP-Rib and NADH in vivo. Ithas been reported that in Arabidopsis plants in re-

Figure 7. Effects of suppression of thePAR reaction on oxidative stress toler-ance. Phenotypes (A), survival rates(B), and chlorophyll contents (C) ofwild-type Arabidopsis plants treatedwith 3-AB, a PARP inhibitor, undernormal conditions and oxidative stress.Two-week-old Arabidopsis plants weregrown on MS medium containing 1%dimethyl sulfoxide with or without 5mM 3-AB and 2 mM PQ for 7 d underlong-day conditions. Data aremeans6SD for three individual experiments (n =3). Different letters indicate significantdifferences (P , 0.05). FW, Freshweight.

Figure 8. Effects of suppression of the PAR reactionon the levels of poly(ADP-Rib), NADH, NAD+, andATP under oxidative stress. Two-week-old Arabidop-sis plants were grown on MS medium containing 1%dimethyl sulfoxide with or without 5 mM 3-AB and3 mM PQ for 7 d under long-day conditions. Thelevels of poly(ADP-Rib) (A), NADH (B), NAD+ (C),and ATP (D) in the plants were determined as de-scribed in “Materials and Methods.” Data aremeans 6 SD for at least three individual experiments(n = 3–6). Different letters indicate significant differ-ences (P , 0.05). FW, Fresh weight.

Ishikawa et al.




sponse to pathogenic infections, AtNUDX7 prefersNADH to ADP-Rib as a physiological substrate (Geet al., 2007). The discrepancy between previous find-ings and our findings here may be due to the differ-ence in action of AtNUDX7 depending on the growthconditions or the type of stress.We reported that in Arabidopsis plants occur mul-

tiple ADP-Rib and/or NADH pyrophosphohydrola-ses (e.g. AtNUDX2, -6, and -10) as well as AtNUDX7(Ogawa et al., 2005, 2008). Judging from the pyrophos-phohydrolase activity toward ADP-Rib and NADH inthe KO-nudx7 plants, it was clear that the majority(53.1%) of the NADH pyrophosphohydrolase in thecrude extract from Arabidopsis was AtNUDX7 (Fig.1). In addition, the expression of AtNUDX7 accountedfor 23.1% of the total ADP-Rib pyrophosphohydrolaseactivity. Notably, the expression of AtNUDX7 wasmarkedly induced by various types of oxidative stress(Fig. 3). We have previously reported that AtNUDX2preferred ADP-Rib to NADH as a physiological sub-strate and that overexpression of AtNUDX2 led to anenhancement of tolerance to oxidative stress caused bytreatment with PQ and salinity by accelerating nu-cleotide recycling from ADP-Rib produced by poly(ADP-Rib) metabolism, leading to suppression of theoverconsumption of NAD+ and ATP in plant cells un-der stressful conditions (Ogawa et al., 2009). However,the total activity of ADP-Rib pyrophosphohydrolase inthe AtNUDX2-knockdown plants was only slightlyreduced (approximately 10%) compared with that inthe control plants, although protein levels of the en-zyme were reduced to less than 30%. These findingsindicated that the expression of endogenous AtNUDX2is very weak and that AtNUDX2 might not contribute

substantially to cellular defense systems in situ. Incontrast, the results obtained here indicate AtNUDX7to be substantially associated with the metabolism ofboth ADP-Rib and NADH in Arabidopsis cells undernormal and stressful conditions.

AtNUDX7 Controls Intracellular NADH Levels inArabidopsis Cells under Normal Conditions

NAD+ is produced via de novo and salvage path-ways. In mature plants, the salvage pathway, wherebynicotinamide is recycled back to NAD+ through nico-tinic acid, contributes most to the NAD(P)(H) pool(Wang and Pichersky, 2007). In addition, a recent studysuggests the presence of a novel salvage pathway;NADH can be directly generated from NMNH andATP by the actions of nicotinamide mononucleotideadenylyl transferases (Berger et al., 2005), althoughthe physiological significance of this third pathwayhas not been established yet. By the reaction withAtNUDX7, NADH was hydrolyzed to NMNH andAMP (Ogawa et al., 2005). Changes of NADH levels inthe control, KO-nudx7, and Pro35S:AtNUDX7 plants(Fig. 2) suggest that AtNUDX7 functions in the turn-over of NADH coupling to the novel salvage pathway.Recently, Hashida et al. (2007) reported that a reduc-tion in the expression of AtNMNAT, a gene encodingnicotinate/nicotinamidemononucleotide adenyltrans-ferase, results in a decrease in NAD(H) content inpollen, inhibition of pollen tube growth, and subse-quent shortening of siliques with lower seed sets,suggesting the importance of NAD(H) accumulationduring the development of pollen. The AtNUDX7mRNA is expressed ubiquitously in all plant tissues

Figure 9. Effects of suppression ofthe PAR reaction on the expressionof genes related to DNA repairsystems. Experimental conditionsare the same as in Figure 8. Quan-titative RT-PCR analysis of thegenes related to DNA repair sys-tems in the plants treated with orwithout 5 mM 3-AB and 3 mM PQwas carried out as described inFigure 6. Relative amounts werenormalized to Actin2 mRNA. Dataare means6 SD for three individualexperiments (n = 3). Different let-ters indicate significant differences(P , 0.05).





(Ogawa et al., 2005). However, there was no differencein growth or morphology between the control plantsand the plants in which AtNUDX7 was overexpressedor disrupted, probably because the altered expressionof AtNUDX7 does not lead to a disturbance in theaccumulation NAD+, which accounts for the majorityof the NAD(P)(H) pool (Fig. 5).

To consider that redox equivalents might rapidlycross different compartments, we analyzed effectsof the overexpression or disruption of AtNUDX7 onthe levels and redox status of primary reductants,AsA and GSH, in plants. However, there was nodifference in the levels of AsA, DAsA, GSH, andGSSG in the control, Pro35S:AtNUDX7, and KO-nudx7plants under both normal conditions and oxidativestress (data not shown). It is well known that most ofthe AsA and GSH in green tissues are found in thechloroplasts (Noctor and Foyer, 1998; Meyer and Hell,2005; Mullineaux and Rausch, 2005; Ishikawa andShigeoka, 2008). Similarly, 30% to 40% of total cellularNADH is in the chloroplast (estimated concentration,0.4 mM), and the remaining 45% to 55% and 10% to15% are in the cytosol (0.65 mM) and the mitochon-drion (2.4 mM), respectively (Wigge et al., 1993).Therefore, it is reasonable that the levels and redoxstates of AsA and GSH are not affected by NADHmetabolism as a result of AtNUDX7 in the cytosol. Asimilar result was observed in mitochondrial complexI-deficient tobacco (Nicotiana tabacum) plants, whichrespire through alternative respiratory dehydroge-nases. These plants showed a 2-fold increase in leafcontents of NAD+ and NADH without an appreciableincrease in NADP+, NADPH, hydrogen peroxide,AsA, or GSH (Dutilleul et al., 2003, 2005).

AtNUDX7 Is Indispensable for Defense againstOxidative Stress

Recently, several researchers have demonstratedthat AtNUDX7 functions in the immune responses topathogens, although doubts remain about the directeffect of AtNUDX7 on the responses (Bartsch et al.,2006; Jambunathan and Mahalingam, 2006; Ge et al.,2007; Adams-Phillips et al., 2008). AtNUDX7 is likelyassociated with defense systems against oxidativestress, since cellular redox status is closely relatednot only to the response to pathogenic infection butalso to various forms of abiotic stress (Foyer andNoctor, 2005). Based on the indispensable roles ofNADH in numerous metabolic reactions, the action ofAtNUDX7 is thought to be disadvantageous in thecells. Therefore, the expression of AtNUDX7 might befinely tuned in response to oxidative stress. In fact, theexpression of AtNUDX7 was temporarily induced inresponse to various stressful conditions (Fig. 3). Sim-ilar expression patterns of AtNUDX7 are observed inthe Genevestigator Arabidopsis microarray database(Zimmermann et al., 2004). According to the informa-tion from the database, it is notable that AtNUDX7 ishighly expressed in response to various types of

oxidative stress, such as high light, UV irradiation,osmotic shock, drought, cold, salinity, and wounding,in addition to pathogen infections and treatmentswith elicitor. Furthermore, the Pro35S:AtNUDX7 plantshaving high levels of AtNUDX7 expression clearlyshowed enhanced tolerance to oxidative stress causedby PQ treatment compared with the control plants(Fig. 4). In contrast, the KO-nudx7 plants showedenhanced sensitivity to the stress. These findings sug-gest that the actions of AtNUDX7 are indispensable forthe systems of defense against oxidative stress.

AtNUDX7 Modulates the PAR Reaction in Response toOxidative Stress

While it has long been thought that the majorcellular functions of NAD+ and NADH are to modu-late cellular energy metabolism in organisms, increas-ing evidence has suggested that NAD+ and NADHalso play key roles in cell death and various majorcellular functions, such as Ca2+ homeostasis and geneexpression (Rutter et al., 2001; Berger et al., 2004). Inparticular, NAD+ has been implicated in the control ofgene expression through the PAR reaction; NAD+

must be continually produced for the normal func-tioning of PARP. Furthermore, Ca2+ appears to be animportant cofactor in PARP’s hyperactivation (Bentleet al., 2006). It was noteworthy that, in the Pro35S:AtNUDX7 and KO-nudx7 plants, the amount of poly(ADP-Rib) correlated with the expression level ofAtNUDX7 (Fig. 5). NADH and NAD+ pass throughthe nuclear envelope (Zhang et al., 2002). These find-ings suggest that AtNUDX7 functions in the modula-tion of the PAR reaction in response to oxidative stresseither directly by the metabolism of NADH in thecytosol or indirectly by perturbation of the NADH/NAD+ ratio or by subsequent disruption of Ca2+ ho-meostasis. Notably, suppression of the PAR reaction bytreatment with 3-AB enhanced the sensitivity to oxi-dative stress (Fig. 7), suggesting that activation of thePAR reaction by AtNUDX7 under oxidative stress was,at least in part, responsible for tolerance to the stress.

Maintenance of NAD+ and ATP Levels by AtNUDX7

under Oxidative Stress

Recently, it has been reported that the expressionof genes encoding PARPs and PARGs in Arabidopsiswas induced by various stressful conditions (Doucet-Chabeaud et al., 2001; Ogawa et al., 2009). Further-more, the activation of poly(ADP-Rib) metabolismreflecting the activation of PARPs occurred in Arabi-dopsis under oxidative stress caused by PQ treatment(Fig. 5; Ogawa et al., 2009). It is well established thathypersynthesis of PAR causes depletion of NAD+ andATP, leading to energy failure and cell necrosis (Loset al., 2002; De Block et al., 2005). Accumulation ofNAD+ and ATP was observed in Arabidopsis plants inwhich the PARP activity was reduced by gene silenc-ing (De Block et al., 2005). Under stressful conditions,

Ishikawa et al.




the activation of the PAR reaction in the control plantscaused decreased levels of NAD+ and ATP (Fig. 5, Band C). The excess activation of the PAR reaction in thePro35S:AtNUDX7 plants resulted in a decrease in ATPlevel even under normal conditions (Figs. 2 and 5). Onthe other hand, NAD+ and ATP were accumulated athigh levels in the KO-nudx7 and 3-AB-treated plants,in which the PAR reaction was suppressed, undernormal and stressful conditions (Figs. 5 and 8). Theseresults clearly indicated that activation of the PARreaction causes depletion of NAD+ and ATP in Arabi-dopsis plants.Free ADP-Rib is produced via a variety of pathways

but is mainly produced via a reversed PAR by PARG(Olivera et al., 1989). Rossi et al. (2002) have reportedthat the ADP-Rib produced from poly(ADP-Rib) israpidly degraded to AMP and that the production ofAMP might be an important pathway to reestablishutilizable energy units. In addition, it has been dem-onstrated that an important function of ATP is derivedfrom poly(ADP-Rib) for the maintenance of the repli-cation apparatus during DNA repair (Maruta et al.,2007). Furthermore, nicotinamide was released by thedegradation of NAD+ by PARP and returned to NAD+

by the salvage pathway, which was accompanied byATP consumption (Noctor et al., 2006). By the action ofAtNUDX7 as an ADP-Rib pyrophosphohydrolase,ADP-Rib is hydrolyzed to AMP and Rib 5-P (Ogawaet al., 2005). It has been demonstrated that adenylatekinase catalyzes a reversible transphosphorylation re-action interconverting ADP to ATP and AMP (Noda,1973; Pradet and Raymond, 1983; Carrari et al., 2005;Lange et al., 2008). In addition, ATP synthase serves tosynthesize ATP from ADP and free phosphate (Wuand Ort, 2008; Zhang et al., 2008). These findings andthe levels of ADP-Rib, NAD+, and ATP in the control,KO-nudx7, and Pro35S:AtNUDX7 plants (Fig. 5) suggestthat, in addition to NAD+ supply coupling to the novelsalvage pathway as described above, AtNUDX7 func-tions in the maintenance of NAD+ by supplying ATPthrough nucleotide recycling from free ADP-Rib mol-ecules under the stress. Consequently, similar to thecase of AtNUDX2 (Ogawa et al., 2009), it was reason-able that the overexpression of AtNUDX7 confersenhanced tolerance to oxidative stress in Arabidopsisplants.

Regulation of the DNA Repair Pathways via Modulationof the PAR Reaction by AtNUDX7 under Oxidative Stress

Cellular DNA, RNA, and their precursor nucleo-tides are at high risk of being oxidized by ROS underoxidative stress (Nakabeppu et al., 2006). PARP detectsSSB generated directly or resulting from the process-ing of damaged bases by the SSBR/BER pathway andactivates the repair reaction (Schreiber et al., 2006). Thelack of PARP rendered cells significantly sensitive toalkylating agent and g-radiation (Trucco et al., 1998).The acute hypersensitivity and the high genomic in-stability in PARP-null mice to alkylation caused dra-

matic decreases in DNA strand-break rejoining (deMurcia et al., 1997). It has been demonstrated thatPARP interacts with an adaptor protein, XRCC1,which has two interfaces with two important SSBR/BER enzymes, DNA ligase III and DNA polymerase b(Caldecott et al., 1995, 1996; Kubota et al., 1996;Masson et al., 1998). In addition, Rad51 is a key factorin homologous recombination for the repair of double-strand DNA breaks (Lees-Miller and Meek, 2003;Dudas and Chovanec, 2004). It has been shown thatthe expression of Rad51 is negatively correlated withPARP activity, since an absence of PARP activityincreases the amount of SSB converted into double-strand DNA breaks (Schultz et al., 2003). Numeroushomologs of E. coli RecA, AtRAD51, AtDMC1,AtRAD51B, AtRAD51C, AtRAD51D, AtXRCC2, andAtXRCC3, were identified in Arabidopsis (Klimyukand Jones, 1997; Doutriaux et al., 1998). Disruption ofArabidopsis XRCC2 (Atxrcc2) conferred hypersensi-tivity to the DNA cross-linking agent mitomycin C(Bleuyard andWhite, 2004). Similarly, Atxrcc3mutantswere hypersensitive to mitomycin C (Bleuyard et al.,2005). These findings clearly indicated that the E. coliRecA-like protein is involved in recombinational re-pair in Arabidopsis.

The expression levels of AtXRCC1 and -2 paralleledthat of AtNUDX7 under both normal conditionsand oxidative stress (Fig. 6). On the other hand, aninverse correlation was observed between the levelsof AtXRCC3, AtRAD51, AtDMC1, and AtMND1 andAtNUDX7. As in the KO-nudx7 plants, the down- orup-regulation of those genes was observed in the3-AB-treated plants (Fig. 9). These findings suggest thatthe expression of those genes encoding factors relatedto the repair of DNA is either positively or negativelyregulated by the PAR reaction in Arabidopsis and thatthe regulation contributes, in part, to the tolerance tooxidative stress. Therefore, it is likely that AtNUDX7is involved in the regulation of defense mechanismsagainst oxidative DNA damage via modulation of thePAR reaction.

MATERIALS AND METHODS

Materials and Plant Growth Conditions

The vectors for the Gateway cloning system, pDONR201 and pGWB2,

were obtained fromDr. Tsuyoshi Nakagawa (Shimane University). Restriction

enzymes and modifying enzymes were purchased from Takara. All other

chemicals were of analytical grade and used without further purification.

Arabidopsis (Arabidopsis thaliana) ecotype Columbia was grown under long-

day conditions (16 h of light, 25�C/8 h of dark, 22�C) onMurashige and Skoog

(MS) medium under a light intensity of 100 mE m22 s21.

Generation of Transgenic Plants

Total RNAwas isolated from the leaves of 4-week-old Arabidopsis plants

(1.0 g fresh weight), as described previously (Yoshimura et al., 1999). First-

strand cDNA was synthesized using ReverTra Ace reverse transcriptase

(Toyobo) with an oligo(dT) primer. The vector for the generation of the

AtNUDX7-overexpressed plants was constructed using Gateway cloning

technology (Invitrogen). The cDNA encoding the open reading frame of





AtNUDX7 was cloned into the donor vector, pDONR201, and then recloned

into the destination vector, pGWB2. The specific primers with attB1 and attB2

sequences were as follows: attB1-AtNUDX7 (5#-AAAAAGCAGGCTATGGG-

TACTAGAGCTCAG-3#) and attB2-AtNUDX7 (5#-AGAAAGCTGGGTTCA-

GAGAGAAGCAGAGGC-3#). PCR and in vitro BP and LR recombination

reactions were carried out according to the manufacturer’s instructions

(Invitrogen).

Agrobacterium tumefaciens, which was transformed with the obtained

constructs by electroporation, was used to infect Arabidopsis via the vacuum

infiltration method. T1 seedlings were selected on basic MS medium in petri

dishes containing 3% Suc, 20 mg L21 hygromycin, and 20 mg L21 kanamycin

for 2 weeks and then transferred to soil. T3 seeds were harvested and used

for the experiments. The knockout Arabidopsis line (KO-nudx7; obtained

through the SIGnAL project [http://signal.salk.edu/]) containing a T-DNA

insert in the AtNUDX7 gene (At4g12720) was outcrossed and selfed to check

for segregation and to obtain a purely homozygous line.

Northern-Blot Analysis

Total RNA (30 mg each) extracted from the leaves of 4-week-old trans-

formants (T3 generation) overexpressing AtNUDX7 was subjected to a

northern-blot analysis as described previously (Yoshimura et al., 2004).

Autoradiography was carried out using a phosphor imager (Mac BAS 1000;

Fuji Photofilm). The transcript levels were estimated from the densitometric

readings of three independent experiments and expressed as relative expres-

sion ratios.

Measurement of Chlorophyll Content

Chlorophyll was extracted with acetone at 4�C from 0.2 g of seedling and

measured by the method of Arnon (1949).

Western-Blot Analysis

A polyclonal mouse antibody against the AtNUDX7 protein was prepared

using His-tagged recombinant AtNUDX7 protein, synthesized as described

previously (Ogawa et al., 2005). Western-blot analysis was carried out as

reported (Yoshimura et al., 2004). The AtNUDX7 protein was detected using

the specific polyclonal antibody as the primary antibody and anti-mouse IgG-

horseradish peroxidase conjugate (Bio-Rad) as the secondary antibody. Pro-

tein bands were detected using the enhanced chemiluminescence detection

system (GE Healthcare). The protein concentration was determined by the

method of Bradford (1976).

Analysis of ADP-Rib and NADHPyrophosphohydrolase Activities

The leaves (0.5 g) of Arabidopsis plants were homogenized with 1 mL of

100 mM Tris-HCl (pH 8.0) containing 20% glycerol. After centrifugation

(20,000g) for 20 min at 4�C, the supernatant was used for analysis of the

enzymatic activity. ADP-Rib and NADH pyrophosphohydrolase activities

were assayed by coupling to alkaline phosphatase and measuring colorimet-

rically the amount of inorganic phosphate formed at 37�C (Ames, 1966;

Ribeiro et al., 2001). The standard assay mixture contained, in a volume of 0.1

mL, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 0.1 mM substrates, 0.7 units of

alkaline phosphatase, 1 mg mL21 bovine serum albumin, and crude extract

(approximately 10.0 mg of protein). The reaction was stopped and color was

developed by addition of 1 mL of standard inorganic phosphate reagent (6

volumes of 3.4 mM ammoniummolybdate in 0.5 M H2SO4, 1 volume of 570 mM

AsA, and 1 volume of 130 mM SDS; Canales et al., 1995; Ribeiro et al., 2001).

Blanks without enzyme and/or substrate were run in parallel. Enzyme

activities were linear with time and amount of enzyme.

Determination of NAD+, NADH, and ATP Contents

NAD+ and NADH were quantified as described previously (Maciejewska

and Kacperska, 1987; Yoshimura et al., 2004; Ogawa et al., 2008). ATP was

quantified using the LL-100-1 ATP assay system (Toyo) as described previ-

ously (Ogawa et al., 2008). Chemiluminescence was detected using a lumino-

meter (AB-2200-R; Atto).

Determination of Antioxidants

AsA, DAsA, GSH, and GSSG levels were measured according to Yoshimura

et al. (2004).

Immunological Detection of PAR Activity

PAR activity was quantified as described (Ogawa et al., 2008). The protein

(7.5 mg) extracted from Arabidopsis leaves was spotted on a polyvinylidene

difluoride membrane (Bio-Rad). The poly(ADP-ribosyl)ated proteins were

detected using anti-PAR antibody (Biomol) and anti-mouse IgG-horseradish

peroxidase conjugate (Bio-Rad) as a secondary antibody. The quantitative

intensity was determined by applying densitometry to video images of the

blots (Atto).

Determination of ADP-Rib

ADP-Rib was quantified as described previously (Ogawa et al., 2008).

Extracted samples were subjected to HPLC with suppressed conductivity

detection in an ICS-3000 system (Dionex). Dionex IonPac AS11 (4 3 250 mm)

and AG11 guard (4 3 50 mm) were used as the separation columns. Fractions

corresponding to the retention time of ADP-Rib (15–20 min) were collected,

lyophilized, resuspended in 20 mL of water, and analyzed by CE-ESI-MS/MS.

CE-ESI-MS/MS analyses were performed on a P/ACE MDQ capillary elec-

trophoresis system (Beckman Coulter) coupled to a 4000QTRAP hybrid triple

quadrupole linear ion-trap mass spectrometer (Applied Biosystems). CE

separations were carried out at 20�C with silica capillaries (80 cm 3 50 mm

i.d.; GL Sciences). An MP-711 microflow pump (GL Sciences) was used to

provide the sheath liquid flow. ESI-MS/MS was conducted in the negative ion

mode. An ion spray voltage of24.5 kVwas applied. The parameters of curtain

gas, collision gas, temperature, ion source gas 1, ion source gas 2, and entrance

potential were 25.0, 5.0, 0.0, 20.0, 0.0, and –10.0, respectively. Multiple reaction

monitoring transitions (mass-to-charge ratio of precursor ion/mass-to-charge

ratio of product ion) for ADP-Rib and PIPES were 558/346 and 301/193,

respectively.

Stress and 3-AB Treatments

Arabidopsis plants were subjected to various forms of stress: treatment

with PQ and salinity, high light, and drought. PQ treatment was imposed by

growing 7-d-old seedlings onMSmedium containing the agent at 2 to 3 mM for

0 to 7 d under normal light (100 mE m22 s 21) or for 0 to 12 h under high light

(1,600 mE m22 s 21). Salinity stress was imposed by growing the plants on MS

medium containing 250 mM NaCl for 0 to 48 h. Drought stress was imposed by

subjecting the plants to dehydration on paper towels for 0 to 6 h.

For inhibition of PARP activity, 2-week-old Arabidopsis plants were

transferred to MS medium containing 1% dimethyl sulfoxide with or without

5 mM 3-AB (Sigma) and 3 mM PQ for 7 d.

Plant survival was calculated from the ratio of plants keeping with green

and growth to all plants tested.

Quantitative RT-PCR Analysis

Total RNA (50 mg) extracted fromArabidopsis leaves was purified with the

RNeasy Plant Mini Kit (Qiagen), then treated with DNase I to eliminate any

DNA contamination (Takara), and was converted into first-strand cDNA

using ReverTra Ace (Toyobo) with the oligo(dT)20 primer. Primer pairs for the

quantitative RT-PCR designed using Primer Express software (Applied Bio-

systems) were as follows: AtNUDX7-F (5#-CTTGGGATTCGCCATTGTG-3#),AtNUDX7-R (5#-CATGATCCGCATTGCAGTAGAT-3#), AtDMC1-F (5#-AGC-

CAGCAGGTGGTCATGTACT-3#), AtDMC1-R (5#-ACTGCGACAATGGTGT-

TCAAAC-3#), AtNMD1-F (5#-ATTCCCGCCTCCGTGTATAGAT-3#), AtNMD1-R

(5#-CGCCTTTGCCTTTCCTGAA-3#), AtRAD51-F (5#-AAACCCAGCACGG-

ACCTTTC-3#), AtRAD51-R (5#-AGCATCCCTAAGCTTCTTTACATCAA-3#),AtRAD51B-F (5#-CCTTCCCGTTCCATATAACATCAG-3#), AtRAD51B-R

(5#-TGATTCCTGGACCTTTCAGTTCA-3#), AtRAD51C-F (5#-CAAAGTTTA-

GTGAAGGCTCGTTTCA-3#), AtRAD51C-R (5#-CTCGGTTGGTGCACGA-

ATG-3#),AtRAD51D-F (5#-CTGGAGACAAAGAGACGGACTCA-3#), AtRAD51D-R(5#-GAGGAAGGTCCGACAAGTTCTGT-3#), AtXRCC1-F (5#-GGATGAAG-

GACCAACCGAAGA-3#), AtXRCC1-R (5#-AACTTGGCTCGGCGTGTTC-3#),AtXRCC2-F (5#-CGCCACTTCACCGTGTACCT-3#), AtXRCC2-R (5#-CGT-

Ishikawa et al.




AGATGCGCCGGTGAT-3#), AtXRCC3-F (5#-TGCAGAAGGATCCGGAG-

ATG-3#), AtXRCC3-R (5#-GGCTGAATTGATCCTCTCGAACT-3#), Actin2-F

(5#-GGCAAGTCATCACGATTGG-3#), and Actin2-R (5#-CAGCTTCCATTCC-

CACAAAC-3#). Quantitative RT-PCR was performed with an Applied Bio-

systems 7300 Real Time PCR System (Applied Biosystems) using the SYBR

Premix ExTaq (Takara). Actin2 mRNAwas used as an internal standard in all

experiments.

Data Analysis

Significance of differences between data sets was evaluated by t test.

Calculations were carried out with Microsoft Excel software.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers At4g12720 (AtNUDX7), At1g80420

(AtXRCC1), At5g64520 (AtXRCC2), At5g57450 (AtXRCC3), At5g20850

(AtRAD51), At2g28560 (AtRAD51B), At2g45280 (AtRAD51C), At1g07745

(AtRAD51D), At3g22880 (AtDMC1), and At4g29170 (AtMND1).

ACKNOWLEDGMENT

We are grateful to Dr. Tsuyoshi Nakagawa for his generous donation of

the pDONR201 and pGWB2 vectors.

Received April 25, 2009; accepted August 3, 2009; published August 5, 2009.

LITERATURE CITED

Adams-Phillips L, Wan J, Tan X, Dunning FM, Meyers BC, Michelmore

RW, Bent AF (2008) Discovery of ADP-ribosylation and other plant

defense pathway elements through expression profiling of four different

Arabidopsis-Pseudomonas R-avr interactions. Mol Plant Microbe Inter-

act 21: 646–657

Ames BN (1966) Assay of inorganic phosphate, total phosphate and

phosphatases. Methods Enzymol 1: 115–118

Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenolox-

idase in Beta vulgaris. Plant Physiol 24: 1–15

Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J,

Parker JE (2006) Salicylic acid-independent ENHANCED DISEASE

SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is

regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7.

Plant Cell 18: 1038–1051

Baxter CJ, Redestig H, Schauer N, Repsilber D, Patil KR, Nielsen J, Liu J,

Fernie AR (2007) The metabolic response of heterotrophic Arabidopsis

cells to oxidative stress. Plant Physiol 143: 312–325

Bentle MS, Reinicke KE, Bey EA, Spitz DR, Boothman DA (2006)

Calcium-dependent modulation of poly(ADP-ribose) polymerase-1 al-

ters cellular metabolism and DNA repair. J Biochem 281: 33684–33696

Berger F, Lau C, Dahlmann M, Ziegler M (2005) Subcellular compartmen-

tation and differential catalytic properties of the three human nicotin-

amide mononucleotide adenylyltransferase isoforms. J Biol Chem 280:

36334–36341

Berger F, Ramırez-Hernandez MH, Ziegler M (2004) The new life of a

centenarian: signalling functions of NAD(P). Trends Biochem Sci 29:

111–118

Bessman MJ, Frick DN, O’Handley SF (1996) The MutT proteins or

“Nudix” hydrolases, a family of versatile, widely distributed, “house-

cleaning” enzymes. J Biol Chem 271: 25059–25062

Bleuyard JY, Gallego ME, Savigny F, White CI (2005) Differing require-

ments for the Arabidopsis Rad51 paralogs in meiosis and DNA repair.

Plant J 41: 533–545

Bleuyard JY, White CI (2004) The Arabidopsis homologue of Xrcc3 plays

an essential role in meiosis. EMBO J 23: 439–449

Bradford M (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding. Anal Biochem 72: 248–254

Caldecott KW, Aoufouchi S, Johnson P, Shall S (1996) XRCC1 polypeptide

interacts with DNA polymerase beta and possibly poly (ADP-ribose)

polymerase, and DNA ligase III is a novel molecular ‘nick-sensor’ in

vitro. Nucleic Acids Res 24: 4387–4394

Caldecott KW, Tucker JD, Stanker LH, Thompson LH (1995) Character-

ization of the XRCC1-DNA ligase III complex in vitro and its absence

from mutant hamster cells. Nucleic Acids Res 23: 4836–4843

Canales J, Pinto RM, Costas MJ, Hernandez MT, Miro A, Bernet D,

Fernandez A, Cameselle JC (1995) Rat liver nucleoside diphosphosugar

or diphosphoalcohol pyrophosphatases different from nucleotide py-

rophosphatase or phosphodiesterase I: substrate specificities of Mg(2+)-

and/or Mn(2+)-dependent hydrolases acting on ADP-ribose. Biochim

Biophys Acta 1246: 167–177

Carrari F, Coll-Garcia D, Schauer N, Lytovchenko A, Palacios-Rojas N,

Balbo I, Rosso M, Fernie AR (2005) Deficiency of a plastidial adenylate

kinase in Arabidopsis results in elevated photosynthetic amino acid

biosynthesis and enhanced growth. Plant Physiol 137: 70–82

Davidovic L, Vodenicharov M, Affar EB, Poirier GG (2001) Importance of

poly (ADP-ribose)glycohydrolase in the control of poly (ADP-ribose)

metabolism. Exp Cell Res 268: 7–13

De Block M, Verduyn C, De Brouwer D, Cornelissen M (2005) Poly (ADP-

ribose) polymerase in plants affects energy homeostasis, cell death and

stress tolerance. Plant J 41: 95–106

de Murcia JM, Niedergang C, Trucco C, Ricoul M, Dutrillaux B, Mark M,

Oliver FJ, MassonM, Dierich A, LeMeurM, et al (1997) Requirement of

poly(ADP-ribose) polymerase in recovery from DNA damage in mice

and in cells. Proc Natl Acad Sci USA 94: 7303–7307

Doucet-Chabeaud G, Godon C, Brutesco C, de Murcia G, Kazmaier M

(2001) Ionising radiation induces the expression of PARP-1 and PARP-2

genes in Arabidopsis. Mol Genet Genomics 265: 954–963

Doutriaux MP, Couteau F, Bergounioux C, White C (1998) Isolation and

characterisation of the RAD51 and DMC1 homologs from Arabidopsis

thaliana. Mol Gen Genet 257: 283–291

Dudas A, Chovanec M (2004) DNA double-strand break repair by homol-

ogous recombination. Mutat Res 566: 131–167

Dutilleul C, Garmier M, Noctor G, Mathieu C, Chetrit P, Foyer CH, De

Paepe R (2003) Leaf mitochondria modulate whole cell redox homeo-

stasis, set antioxidant capacity, and determine stress resistance through

altered signaling and diurnal regulation. Plant Cell 15: 1212–1226

Dutilleul C, Lelarge C, Prioul JL, De Paepe R, Foyer CH, Noctor G (2005)

Mitochondria-driven changes in leaf NAD status exert a crucial influ-

ence on the control of nitrate assimilation and the integration of carbon

and nitrogen metabolism. Plant Physiol 139: 64–78

Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a

metabolic interface between stress perception and physiological re-

sponses. Plant Cell 17: 1866–1875

Ge X, Li GJ, Wang SB, Zhu H, Zhu T, Wang X, Xia Y (2007) AtNUDT7, a

negative regulator of basal immunity in Arabidopsis, modulates two

distinct defense response pathways and is involved in maintaining

redox homeostasis. Plant Physiol 145: 204–215

Gutteridge JM, Halliwell B (1989) Iron toxicity and oxygen radicals.

Baillieres Clin Haematol 2: 195–256

Ha HC, Snyder SH (1999) Poly (ADP-ribose) polymerase is a mediator of

necrotic cell death by ATP depletion. Proc Natl Acad Sci USA 96: 13978–

13982

Hashida SN, Takahashi H, Kawai-Yamada M, Uchimiya H (2007) Arabi-

dopsis thaliana nicotinate/nicotinamide mononucleotide adenyltrans-

ferase (AtNMNAT) is required for pollen tube growth. Plant J 49:

694–703

Ishikawa T, Shigeoka S (2008) Recent advances in ascorbate biosynthesis

and the physiological significance of ascorbate peroxidase in photo-

synthesizing organisms. Biosci Biotechnol Biochem 72: 1143–1154

Jagtap P, Szabo C (2005) Poly(ADP-ribose) polymerase and the therapeutic

effects of its inhibitors. Nat Rev Drug Discov 4: 421–440

Jambunathan N, Mahalingam R (2006) Analysis of Arabidopsis Growth

Factor Gene 1 (GFG1) encoding a nudix hydrolase during oxidative

signaling. Planta 224: 1–11

Klimyuk VI, Jones JD (1997) AtDMC1, the Arabidopsis homologue of the

yeast DMC1 gene: characterization, transposon-induced allelic varia-

tion and meiosis-associated expression. Plant J 11: 1–14

Kraszewska E (2008) The plant Nudix hydrolase family. Acta Biochim Pol

55: 663–671

Kroj T, Rudd JJ, Nurnberger T, Gabler Y, Lee J, Scheel D (2003) Mitogen-

activated protein kinases play an essential role in oxidative burst-

independent expression of pathogenesis-related genes in parsley. J Biol

Chem 278: 2256–2264

Kubota Y, Nash RA, Klungland A, Schar P, Barnes DE, Lindahl T (1996)





Reconstitution of DNA base excision-repair with purified human pro-

teins: interaction between DNA polymerase beta and the XRCC1 pro-

tein. EMBO J 15: 6662–6670

Kwak JM, Nguyen V, Schroeder JI (2006) The role of reactive oxygen

species in hormonal responses. Plant Physiol 141: 323–329

Lange PR, Geserick C, Tischendorf G, Zrenner R (2008) Functions

of chloroplastic adenylate kinases in Arabidopsis. Plant Physiol 146:

492–504

Lees-Miller SP, Meek K (2003) Repair of DNA double strand breaks by

non-homologous end joining. Biochimie 85: 1161–1173

Los M, Mozoluk M, Ferrari D, Stepczynska A, Stroh C, Renz A, Herceg Z,

Wang ZQ, Schulze-Osthoff K (2002) Activation and caspase-mediated

inhibition of PARP: a molecular switch between fibroblast necrosis and

apoptosis in death receptor signaling. Mol Biol Cell 13: 978–988

Maciejewska U, Kacperska A (1987) Changes in the level of oxidized and

reduced pyridine nucleotides during cold acclimation of winter rape

plants. Physiol Plant 69: 687–691

Mahalingam R, Gomez-Buitrago A, Eckardt N, Shah N, Guevara-Garcia

A, Day P, Raina R, Fedoroff NV (2003) Characterizing the stress/

defense transcriptome of Arabidopsis. Genome Biol 4: R20

Maruta H, Okita N, Takasawa R, Uchiumi F, Hatano T, Tanuma S (2007)

The involvement of ATP produced via (ADP-ribose)n in the mainte-

nance of DNA replication apparatus during DNA repair. Biol Pharm

Bull 30: 447–450

Masson M, Niedergang C, Schreiber V, Muller S, Menissier-de Murcia J,

de Murcia G (1998) XRCC1 is specifically associated with poly(ADP-

ribose) polymerase and negatively regulates its activity following DNA

damage. Mol Cell Biol 18: 3563–3571

Meyer AJ, Hell R (2005) Glutathione homeostasis and redox-regulation by

sulfhydryl groups. Photosynth Res 86: 435–457

Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends

Plant Sci 7: 405–410

Mullineaux P, Karpinski S (2002) Signal transduction in response to excess

light: getting out of the chloroplast. Curr Opin Plant Biol 5: 43–48

Mullineaux PM, Rausch T (2005) Glutathione, photosynthesis and the

redox regulation of stress-responsive gene expression. Photosynth Res

86: 459–474

Nakabeppu Y, Sakumi K, Sakamoto K, Tsuchimoto D, Tsuzuki T,

Nakatsu Y (2006) Mutagenesis and carcinogenesis caused by the oxi-

dation of nucleic acids. Biol Chem 387: 373–379

Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active

oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:

249–279

Noctor G, Queval G, Gakiere B (2006) NAD(P) synthesis and pyridine

nucleotide cycling in plants and their potential importance in stress

conditions. J Exp Bot 57: 1603–1620

Noda LH (1973) Adenylate kinase. In PD Boyer, ed, The Enzymes (Group

Transfer Part A), Vol VII. Academic Press, New York, pp 279–305

Ogawa T, Ishikawa K, Harada K, Fukusaki E, Yoshimura K, Shigeoka S

(2009) Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2,

confers enhanced tolerance to oxidative stress on Arabidopsis plants.

Plant J 57: 289–301

Ogawa T, Ueda Y, Yoshimura K, Shigeoka S (2005) Comprehensive

analysis of cytosolic Nudix hydrolases in Arabidopsis thaliana. J Biol

Chem 280: 25277–25283

Ogawa T, Yoshimura K, Miyake H, Ishikawa K, Ito D, Tanabe N,

Shigeoka S (2008) Molecular characterization of organelle-type Nudix

hydrolases in Arabidopsis. Plant Physiol 148: 1412–1424

Olivera BM, Hughes KT, Cordray P, Roth JR (1989) Aspects of NAD

metabolism in prokaryotes and eukaryotes. In MK Jacobson, EL Jacobson,

eds, ADP-Ribose Transfer Reactions: Mechanisms and Biological Signifi-

cance, Ed 2, Vol 3. Springer Verlag, New York, pp 353–360

Pradet A, Raymond P (1983) Adenine-nucleotide ratios and adenylate

energy charge in energy metabolism. Annu Rev Plant Physiol Plant Mol

Biol 34: 199–224

Qin XJ, Hudson LG, Liu W, Timmins GS, Liu KJ (2008) Low concentration

of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting

PARP-1 activity. Toxicol Appl Pharmacol 232: 41–50

Ribeiro JM, Carloto A, Costas MJ, Cameselle JC (2001) Human placenta

hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase

and a specific ADP-ribose pyrophosphatase. Biochim Biophys Acta

1526: 86–94

Rossi L, Denegri M, Torti M, Poirier GG, Ivana Scovassi A (2002) Poly

(ADP-ribose) degradation by post-nuclear extracts from human cells.

Biochimie 84: 1229–1235

Rutter J, Reick M, Wu LC, McKnight SL (2001) Regulation of clock and

NPAS2 DNA binding by the redox state of NAD cofactors. Science 293:

510–514

Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Poly(ADP-ribose):

novel functions for an old molecule. Nat Rev Mol Cell Biol 7: 517–528

Schultz N, Lopez E, Saleh-Gohari N, Helleday T (2003) Poly(ADP-ribose)

polymerase (PARP-1) has a controlling role in homologous recombina-

tion. Nucleic Acids Res 31: 4959–4964

Thompson LH, West MG (2000) XRCC1 keeps DNA from getting stranded.

Mutat Res 16: 1–18

Torres MA, Jones JD, Dangl JL (2006) Reactive oxygen species signaling in

response to pathogens. Plant Physiol 141: 373–378

Trucco C, Oliver FJ, de Murcia G, Menissier-de Murcia J (1998) DNA

repair defect in poly(ADP-ribose) polymerase-deficient cell lines. Nu-

cleic Acids Res 26: 2644–2649

Virag L, Szabo C (2002) The therapeutic potential of poly (ADP-ribose)

polymerase inhibitors. Pharmacol Rev 54: 375–429

Wang G, Pichersky E (2007) Nicotinamidase participates in the salvage

pathway of NAD biosynthesis in Arabidopsis. Plant J 49: 1020–1029

Wigge B, Kromer S, Gardestrom P (1993) The redox levels and subcellular

distribution of pyridine nucleotides in illuminated barley leaf proto-

plasts studied by rapid fractionation. Physiol Plant 88: 10–18

Winters TA, Russell PS, Kohli M, Dar ME, Neumann RD, Jorgensen TJ

(1999) Determination of human DNA polymerase utilization for the

repair of a model ionizing radiation-induced DNA strand break lesion

in a defined vector substrate. Nucleic Acids Res 27: 2423–2433

Wu G, Ort DR (2008) Mutation in the cysteine bridge domain of the

gamma-subunit affects light regulation of the ATP synthase but not

photosynthesis or growth in Arabidopsis. Photosynth Res 97: 185–193

Xu W, Dunn CA, Jones CR, D’Souza G, Bessman MJ (2004) The 26 Nudix

hydrolases of Bacillus cereus, a close relative of Bacillus anthracis.

J Biochem 279: 24861–24865

Yoshimura K, Miyao K, Gaber A, Takeda T, Kanaboshi H, Miyasaka H,

Shigeoka S (2004) Enhancement of stress tolerance in transgenic to-

bacco plants overexpressing Chlamydomonas glutathione peroxidase in

chloroplasts or cytosol. Plant J 37: 21–33

Yoshimura K, Yabuta Y, Tamoi M, Ishikawa T, Shigeoka S (1999) Alter-

natively spliced mRNA variants of chloroplast ascorbate peroxidase

isoenzymes in spinach leaves. Biochem J 338: 41–48

Zhang Q, Piston DW, Goodman RH (2002) Regulation of corepressor

function by nuclear NADH. Science 295: 1895–1897

Zhang X, Liu S, Takano T (2008) Overexpression of a mitochondrial ATP

synthase small subunit gene (AtMtATP6) confers tolerance to several

abiotic stresses in Saccharomyces cerevisiae and Arabidopsis thaliana.

Biotechnol Lett 30: 1289–1294

Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004)

GENEVESTIGATOR: Arabidopsis microarray database and analysis

toolbox. Plant Physiol 136: 2621–2632

Ishikawa et al.




modulation of the poly(adp-ribosyl)ation reaction via the

Documents