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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Modulation of the Poly(ADP-ribosyl)ation Reaction via the Arabidopsis ADP-Ribose/NADH Pyrophosphohydrolase, AtNUDX7, Is Involved in the Response to Oxidative Stress^1,[OA]

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, Osaka 565–0871, Japan (E.H., Y.N., K.H., E.F.); and Department of Food and Nutritional Science, College of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi 487–8501, Japan (K.Y.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 (Pro_35S:AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro_35S:AtNUDX7 plants but increased in the KO-nudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro_35S:AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro_35S: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 completely suppressed in the Pro_35S:AtNUDX7 plants. Accumulation of NAD⁺ and ATP was observed in the KO-nudx7- and 3-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 both normal 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 nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD⁺ by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.

Among various defense systems against attack by ROS, the poly(ADP-ribosyl)ation (PAR) of proteins by poly(ADP-Rib)polymerase (PARP), by which branched polymers of ADP-Rib are attached using β-NAD⁺ to a specific amino acid residue of an acceptor protein, is a posttranslational modification for responding early to DNA damage, such as single-strand DNA break and resealing, caused by oxidative stress and, thus, is crucial for genomic integrity and cell survival (Qin et al., 2008). PARP detects DNA strand breaks and converts the damage into intracellular signals that can activate DNA repair programs or cell death, according to the severity of the injury, via the PAR reaction of nuclear proteins involved in chromatin architecture and DNA metabolism and interacts with the x-ray repair cross complementing factor 1 (XRCC1), an adaptor protein that also has two interfaces with two important single-strand DNA break (SSB) repair (SSBR)/base excision repair (BER) enzymes: DNA ligase and DNA polymerase β (Caldecott et al., 1995, 1996; Kubota et al., 1996; Masson et al., 1998). DNA polymerase β fills the single nucleotide gap, preparing the strand for ligation by a complex of DNA ligase III and XRCC1 (Winters et al., 1999; Thompson and West, 2000). Thereby, the fast recruitment of SSBR/BER factors is archived in the site of the lesion. Modifications of proteins with poly(ADP-Rib) are reversed by poly(ADP-Rib) glycohydrolase (PARG), by which ADP-Rib polymers are hydrolyzed to free ADP-Rib, since incorrect signal transduction is caused by excessive accumulation of poly(ADP-Rib) modification (Davidovic et al., 2001). However, it has been reported that a massive PAR reaction results in the overconsumption of NAD⁺ and ATP and, ultimately, in energy depletion causing necrotic cell death (Ha and Snyder, 1999; Virág and Szabó, 2002; De Block et al., 2005).

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases catalyze the hydrolysis of intact and oxidatively damaged nucleoside diphosphates and triphosphates, nucleotide sugars, coenzymes, dinucleoside polyphosphates, and RNA caps in various organisms such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). We have previously reported the characteristics of cytosolic Nudix hydrolases (AtNUDX1–AtNUDX11) in Arabidopsis (Arabidopsis thaliana; Ogawa et al., 2005). Among them, the recombinant AtNUDX7 showed high affinity for ADP-Rib and NADH as substrates in vitro, converting NADH to a reduced form of nicotinamide mononucleotide (NMNH) plus AMP and ADP-Rib to AMP plus Rib 5-P (Ogawa et al., 2005). AtNUDX7 was expressed more strongly in leaf than in stem and root. Therefore, the enzyme might be involved in nucleotide recycling relating to the metabolism of NADH and/or poly(ADP-Rib).

Recent studies revealed that the actions of AtNUDX7 (At4g12720) are closely related to immune responses to pathogens. Knockout of AtNUDX7 (KO-nudx7) in Arabidopsis plants led to deleterious inference for cells, such as microscopic cell death, constitutive expression of pathogenesis-related genes, resistance to bacterial pathogens, and accumulation of NADH (Jambunathan and Mahalingam, 2006). Furthermore, AtNUDX7 exerted a negative regulatory effect on EDS1 signaling, which controls the activation of defenses and programmed cell death conditioned by intracellular Toll-related immune receptors that recognized specific pathogen effectors (Bartsch et al., 2006). More recently, Ge et al. (2007) reported that KO-nudx7 plants show heightened defense responses, which are both dependent on and independent of the accumulation of NPR1 and salicylic acid, to pathogenic attack. On the other hand, Adams-Phillips et al. (2008) reported that KO-nudx7 plants exhibit a reduced hypersensitive-response phenotype, although the growth of both virulent and avirulent pathogens is suppressed in the plants. These findings support the hypothesis that regulation of the metabolism of NADH and/or ADP-Rib by Nudix hydrolases is important for stress-related defense systems in higher plants. However, the direct actions of the enzymes on stress responses are not established yet.

In this study, to assess the functions of Arabidopsis Nudix hydrolases having ADP-Rib and NADH pyrophosphohydrolase activities under normal conditions and oxidative stress, we analyzed the effect of the overexpression or disruption of AtNUDX7 on levels of ADP-Rib, NAD(H), and ATP as well as PAR activity and oxidative stress tolerance in Arabidopsis. The evidence presented here suggests that AtNUDX7 serves to balance between NADH and NAD⁺ by NADH turnover under normal conditions. In addition, AtNUDX7 functions in the maintenance of NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and the modulation of the PAR reaction, thereby regulating the DNA repair pathways, in response to oxidative stress.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Characteristics of AtNUDX7-Overexpressed or -Disrupted Arabidopsis Plants

We have previously demonstrated that the AtNUDX7 mRNA is expressed ubiquitously in all plant tissues (Ogawa et al., 2005). As shown in Figure 1B , the mRNA and protein (31.8 kD) of AtNUDX7 in the leaves of the wild-type Arabidopsis plants were detected by semiquantitative reverse transcription (RT)-PCR and western-blot analysis, respectively. The pyrophosphohydrolase activities toward ADP-Rib and NADH in the crude extracts prepared from Arabidopsis leaves were 63.2 ± 1.7 and 95.6 ± 3.0 nmol min^–1 mg^–1 protein, respectively (Fig. 1D), consistent with the results reported by Ogawa et al. (2009).

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Characteristics of AtNUDX7-overexpressed or -disrupted Arabidopsis plants. A, T-DNA insertion site in the Arabidopsis 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 plants were grown on MS medium for 2 weeks under long-day conditions and then used for the analysis. Semiquantitative RT-PCR analysis was performed using specific primers for AtNUDX7 and Actin2 in a reaction involving 27 cycles of 95°C for 60 s, 55°C for 60 s, and 72°C for 60 s, followed by 72°C for 10 min. Equal loading of each amplified cDNA was determined with the control Actin2 PCR product. The AtNUDX7 protein was detected by western blotting using a specific polyclonal antibody raised against the recombinant AtNUDX7 protein. C, Northern-blot (top) and western-blot (bottom) analyses of the AtNUDX7 protein in control (transformed with the empty vector) and Pro35S:AtNUDX7 plants. The control and Pro35S:AtNUDX7 (T3 generations of independent 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 cDNA fragment as a probe. Equal loading of each RNA was determined with the Actin2 mRNA. D, ADP-Rib and NADH pyrophosphohydrolase activities in the leaves of wild-type, control, Pro35S:AtNUDX7, and KO-nudx7 plants. Data are means ± SD for three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05).

Next, we generated transgenic Arabidopsis plants overexpressing AtNUDX7 under the control of the cauliflower mosaic virus 35S promoter (Pro_35S:AtNUDX7). Northern-blot analysis showed that the levels of the AtNUDX7 transcript in the T3 generation of Pro_35S:AtNUDX7-5-1, -5-2, and -7-1 plants are approximately 3.7-, 5.6-, and 1.7-fold higher, respectively, than levels in the control plants (transformed with the empty vector; Fig. 1C). By western-blot analysis, a large amount of the AtNUDX7 protein was detected in the extracts prepared from the leaves of Pro_35S:AtNUDX7-5-1, -5-2, and -7-1 plants (Fig. 1C). The levels of protein in the transgenic plants were well correlated with the levels of the transcript. The pyrophosphohydrolase activities toward ADP-Rib and NADH in the Pro_35S:AtNUDX7 plants were approximately 1.2- to 2.5-fold and 1.2- to 2.0-fold, respectively, higher than levels in the control plants (Fig. 1D). No difference was observed in growth or morphology between the control and these transgenic plants under normal conditions (data not shown).

Physiological Substrates for AtNUDX7

We have demonstrated that the recombinant AtNUDX7 protein hydrolyzes both ADP-Rib and NADH in vitro (Ogawa et al., 2005). To clarify the physiological substrate for AtNUDX7 in situ, we analyzed the intercellular levels of ADP-Rib and NADH in Pro_35S:AtNUDX7 and KO-nudx7 plants under normal conditions. NADH accumulated to higher levels in the KO-nudx7 plants than in the control plants (Fig. 2A ). In contrast, the amounts of NADH in the Pro_35S:AtNUDX7 plants were remarkably decreased compared with those in the control plants. According to capillary electrophoresis-electrospray-tandem mass spectrometry (CE-ESI-MS/MS) analysis, the levels of free ADP-Rib in the KO-nudx7 and the Pro_35S:AtNUDX7 plants were significantly higher and lower, respectively, than those in the control plants (Fig. 2B).

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. The levels of intracellular NADH and ADP-Rib under normal conditions and oxidative stress. Two-week-old Arabidopsis plants were grown on MS medium containing 3 µM PQ for 7 d under long-day conditions. 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 ± SD for at least three individual experiments (n = 3–9). Different letters indicate significant differences (P < 0.05). FW, Fresh weight.

To assess the involvement of AtNUDX7 in defense systems against various types of stress, we analyzed the changes in the expression levels of AtNUDX7 by quantitative RT-PCR under various stressful conditions (Fig. 3A ). We have verified that expression of PARP and PARG is induced under the stressful conditions analyzed here (Ogawa et al., 2009). The expression of AtNUDX7 was induced rapidly under conditions of high-light illumination (1,600 µE m^–2 s ^–1), drought (dehydration on a paper towel), salinity (250 mM NaCl), and by treatment with 3 µM paraquat (PQ; an agent producing O₂^–) under high-light illumination, suggesting the participation of excess ROS in the expression. The manipulation for transferring plants in the stress treatments had no effect on the AtNUDX7 expression (data not shown). Western-blot analysis showed that the levels of AtNUDX7 protein were increased by the treatment with PQ (Fig. 3B). Similarly, the pyrophosphohydrolase activities toward both ADP-Rib and NADH in the plant extracts increased by treatment with PQ (Fig. 3C).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Changes in the expression levels of AtNUDX7 under various types of stress. A, Quantitative RT-PCR analysis of AtNUDX7 expression in response to oxidative stress. Two-week-old Arabidopsis plants were subjected to various forms of stress: treatment with PQ (3 µM under illumination at 1,600 µE m–2 s –1), salinity (250 mM NaCl), high-light intensity (1,600 µE m–2 s –1), and drought (dehydration on a paper towel). Total RNA extracted from Arabidopsis leaves was converted into first-strand cDNA using the oligo(dT)20 primer. A quantitative RT-PCR analysis was carried out to determine the expression level of AtNUDX7. The relative amounts were normalized to Actin2 mRNA. Data are means ± 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 were grown on MS medium containing 3 µM PQ under illumination at 1,600 µE m–2 s –1 for 3 h. C, ADP-Rib and NADH pyrophosphohydrolase activities in the leaves of Arabidopsis plants under the PQ treatment. Data are means ± SD for three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05).

Next, we evaluated the contribution of AtNUDX7 to oxidative stress tolerance using Arabidopsis plants in which AtNUDX7 was overexpressed or disrupted. In order to show significant differences in stress tolerance between transgenic and control Arabidopsis plants, the oxidative stress by treatment with 3 µM PQ under normal light (100 µE m^–2 s ^–1) was performed in Pro_35S:AtNUDX7 plants, because the stress by treatment with 3 µM PQ under high light (1,600 µE m^–2 s ^–1) for the analysis of AtNUDX7 expression caused readily severe oxidative damage to plants. As assessed by phenotype, chlorophyll content, and survival rate, the Pro_35S:AtNUDX7-5-1 and -5-2 plants having high expression levels of AtNUDX7 clearly showed enhanced tolerance to the oxidative stress compared with the control plants (Fig. 4, A–C ). Since the 3-aminobenzamide (3-AB)-treated and KO-nudx7 plants were more sensitive to oxidative stress than the control plants, stressful conditions by treatment with 2 µM PQ under normal light (100 µE m^–2 s ^–1) were used for the evaluation of oxidative stress tolerance. In contrast to the Pro_35S:AtNUDX7 plants, the KO-nudx7 plants showed enhanced sensitivity to the stress, while no destruction of chlorophyll was observed in the leaves of wild-type plants after exposure to the stress (Fig. 4, D–F).

View larger version (82K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effects of overexpression or disruption of AtNUDX7 on oxidative stress tolerance. A, Phenotypes of the control and Pro35S:AtNUDX7 plants after oxidative stress caused by PQ treatment. Seven-day-old seedlings were grown on MS medium containing 3 µM PQ for 7 d under long-day conditions. The seedlings were grown then on MS medium without PQ for an additional 7 d. B, Survival rates of the control and Pro35S:AtNUDX7 plants under the PQ treatment. C, Chlorophyll contents of the control and Pro35S:AtNUDX7 plants under normal conditions and oxidative stress. Data are means ± SD for three individual experiments (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 medium containing 2 µM PQ for 7 d under long-day conditions. The plants were grown then on MS medium without PQ for an additional 7 d. E, Survival rates of wild-type and KO-nudx7 plants under PQ treatment. F, Chlorophyll contents of wild-type and KO-nudx7 plants under normal conditions and oxidative stress. Data are means ± SD for three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05). FW, Fresh weight.

Levels of NADH in the KO-nudx7 and Pro_35S:AtNUDX7 plants were significantly high and low, respectively, under stressful conditions (Fig. 2A). The levels of free ADP-Rib in the Pro_35S:AtNUDX7 plants were markedly low compared with those in the control plants under stressful conditions (Fig. 2B). The levels of free ADP-Rib in the KO-nudx7 plants were significantly high under stressful conditions.

Next, we analyzed the amount of poly(ADP-Rib), reflecting the degree of the PAR reaction, in the Pro_35S:AtNUDX7 and KO-nudx7 plants under oxidative stress caused by the treatment with 3 µM PQ. The amount of poly(ADP-Rib) in the control plants was increased under stressful conditions. The amount in the Pro_35S:AtNUDX7 and KO-nudx7 plants was considerably larger and smaller, respectively, than that in the control plants under normal and stressful conditions (Fig. 5A ), indicating a positive correlation between the expression levels of AtNUDX7 and the levels of the PAR reaction.

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. The levels of poly(ADP-Rib), NAD+, and ATP under normal conditions and oxidative stress. Experimental conditions are the same as in Figure 2. The levels of poly(ADP-Rib) (A), NAD+ (B), and ATP (C) in the control, KO-nudx7, and Pro35S:AtNUDX7 plants were determined as described in "Materials and Methods." Data are means ± SD for at least three individual experiments (n = 3–6). Different letters indicate significant differences (P < 0.05). FW, Fresh weight.

The redox homeostasis including antioxidative status in the plant cells is enormously affected by the intracellular NADH level. To obtain more information on the relationship between the cellular redox state and the actions of ADP-Rib/NADH pyrophosphohydrolases, we determined levels of ascorbate (AsA) and glutathione (GSH), as important antioxidants, in the plants in which AtNUDX7 was overexpressed or disrupted. However, the levels of AsA, oxidized AsA (dehydroascorbate [DAsA]), GSH, and oxidized GSH (GSSG) in the Pro_35S:AtNUDX7 and KO-nudx7 plants were unaltered compared with those in the control plants under both normal conditions and oxidative stress caused by the treatment with 3 µM PQ (100 µE m^–2 s ^–1; data not shown).

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

Since activation of the PAR reaction is involved in the repair of DNA damaged by oxidative stress, we analyzed the effect of the overexpression or depletion of AtNUDX7 on the expression of the genes encoding factors involved in the repair: the x-ray repair cross-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 AtXRCC1 and -2 were significantly increased and decreased in the Pro_35S:AtNUDX7 and KO-nudx7 plants, respectively, compared with those in the control plants under both normal conditions and oxidative stress. On the other hand, the levels of AtXRCC3, AtRAD51, AtDMC1, and AtMND1 in the Pro_35S:AtNUDX7 and KO-nudx7 plants were lower and higher, respectively, than those in the control plants under normal conditions or oxidative stress. There was no difference in the levels of AtRAD51B, AtRAD51C, and AtRAD51D between the controls and transformants (data not shown).

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Changes in the expression of genes related to DNA repair systems via modulation of the PAR reaction by AtNUDX7. Experimental conditions are the same as in Figure 2. Quantitative RT-PCR analysis was carried out to determine the expression levels of genes encoding the 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 to Actin2 mRNA. Data are means ± SD for three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05).

Next, we evaluated the effect of suppression of the PAR reaction on oxidative stress tolerance of Arabidopsis plants. It has been reported that 3-AB, a commercially available inhibitor, is used successfully to suppress the PARP activity in plants (Jagtap and Szabo, 2005). Phenotype, chlorophyll content, and survival rate of Arabidopsis plants showed that treatment with 3-AB enhanced the sensitivity to oxidative stress caused by PQ treatment under normal light conditions (Fig. 7 ). The amount of poly(ADP-Rib) in the wild-type Arabidopsis plants treated with 3-AB was significantly smaller than that in the untreated wild-type plants under normal conditions and oxidative stress (Fig. 8A ). Furthermore, NAD⁺ accumulated to higher levels in the 3-AB-treated plants than in the untreated plants under normal and stressful conditions, although the NADH levels in the 3-AB-treated plants were similar to those in the untreated plants (Fig. 8, B and C). The levels of ATP in the 3-AB-treated plants were similar to those in the untreated plants under normal conditions and slightly decreased under stressful conditions (Fig. 8D). Notably, the 3-AB-treated plants showed enhanced sensitivity to oxidative stress compared with the untreated plants (Fig. 7). The expression levels of AtXRCC1 and -2 were significantly decreased in the 3-AB-treated plants compared with those in the untreated plants under both normal conditions and oxidative stress (Fig. 9 ). On the other hand, the levels of AtXRCC3, AtRAD51, AtDMC1, and AtMND1 in the 3-AB-treated plants were higher than those in the untreated plants under normal conditions or oxidative stress.

View larger version (65K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Effects of suppression of the PAR reaction on oxidative stress tolerance. Phenotypes (A), survival rates (B), and chlorophyll contents (C) of wild-type Arabidopsis plants treated with 3-AB, a PARP inhibitor, under normal conditions and oxidative stress. Two-week-old Arabidopsis plants were grown on MS medium containing 1% dimethyl sulfoxide with or without 5 mM 3-AB and 2 µM PQ for 7 d under long-day conditions. Data are means ± SD for three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05). FW, Fresh weight.

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Effects of suppression of the PAR reaction on the levels of poly(ADP-Rib), NADH, NAD+, and ATP under oxidative stress. Two-week-old Arabidopsis plants were grown on MS medium containing 1% dimethyl sulfoxide with or without 5 mM 3-AB and 3 µM PQ for 7 d under long-day conditions. The levels of poly(ADP-Rib) (A), NADH (B), NAD+ (C), and ATP (D) in the plants were determined as described in "Materials and Methods." Data are means ± SD for at least three individual experiments (n = 3–6). Different letters indicate significant differences (P < 0.05). FW, Fresh weight.

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Effects of suppression of the PAR reaction on the expression of genes related to DNA repair systems. Experimental conditions are the same as in Figure 8. Quantitative RT-PCR analysis of the genes related to DNA repair systems in the plants treated with or without 5 mM 3-AB and 3 µM PQ was carried out as described in Figure 6. Relative amounts were normalized to Actin2 mRNA. Data are means ± SD for three individual experiments (n = 3). Different letters indicate significant differences (P < 0.05).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

First, to clarify the physiological substrate for a cytosolic Nudix hydrolase, AtNUDX7, in situ, we analyzed the intercellular levels of NADH and ADP-Rib in Pro_35S:AtNUDX7 and KO-nudx7 plants under normal conditions and oxidative stress (Fig. 2). Our findings strongly suggest that AtNUDX7 functions in the hydrolysis of both ADP-Rib and NADH in vivo. It has been reported that in Arabidopsis plants in response to pathogenic infections, AtNUDX7 prefers NADH to ADP-Rib as a physiological substrate (Ge et al., 2007). The discrepancy between previous findings and our findings here may be due to the difference in action of AtNUDX7 depending on the growth conditions or the type of stress.

We reported that in Arabidopsis plants occur multiple ADP-Rib and/or NADH pyrophosphohydrolases (e.g. AtNUDX2, -6, and -10) as well as AtNUDX7 (Ogawa et al., 2005, 2008). Judging from the pyrophosphohydrolase activity toward ADP-Rib and NADH in the KO-nudx7 plants, it was clear that the majority (53.1%) of the NADH pyrophosphohydrolase in the crude extract from Arabidopsis was AtNUDX7 (Fig. 1). In addition, the expression of AtNUDX7 accounted for 23.1% of the total ADP-Rib pyrophosphohydrolase activity. Notably, the expression of AtNUDX7 was markedly induced by various types of oxidative stress (Fig. 3). We have previously reported that AtNUDX2 preferred ADP-Rib to NADH as a physiological substrate and that overexpression of AtNUDX2 led to an enhancement of tolerance to oxidative stress caused by treatment with PQ and salinity by accelerating nucleotide recycling from ADP-Rib produced by poly(ADP-Rib) metabolism, leading to suppression of the overconsumption of NAD⁺ and ATP in plant cells under stressful conditions (Ogawa et al., 2009). However, the total activity of ADP-Rib pyrophosphohydrolase in the AtNUDX2-knockdown plants was only slightly reduced (approximately 10%) compared with that in the control plants, although protein levels of the enzyme were reduced to less than 30%. These findings indicated that the expression of endogenous AtNUDX2 is very weak and that AtNUDX2 might not contribute substantially to cellular defense systems in situ. In contrast, the results obtained here indicate AtNUDX7 to be substantially associated with the metabolism of both ADP-Rib and NADH in Arabidopsis cells under normal and stressful conditions.

AtNUDX7 Controls Intracellular NADH Levels in Arabidopsis Cells under Normal Conditions

NAD⁺ is produced via de novo and salvage pathways. In mature plants, the salvage pathway, whereby nicotinamide is recycled back to NAD⁺ through nicotinic acid, contributes most to the NAD(P)(H) pool (Wang and Pichersky, 2007). In addition, a recent study suggests the presence of a novel salvage pathway; NADH can be directly generated from NMNH and ATP by the actions of nicotinamide mononucleotide adenylyl transferases (Berger et al., 2005), although the physiological significance of this third pathway has not been established yet. By the reaction with AtNUDX7, NADH was hydrolyzed to NMNH and AMP (Ogawa et al., 2005). Changes of NADH levels in the control, KO-nudx7, and Pro_35S:AtNUDX7 plants (Fig. 2) suggest that AtNUDX7 functions in the turnover of NADH coupling to the novel salvage pathway. Recently, Hashida et al. (2007) reported that a reduction in the expression of AtNMNAT, a gene encoding nicotinate/nicotinamide mononucleotide adenyltransferase, results in a decrease in NAD(H) content in pollen, inhibition of pollen tube growth, and subsequent shortening of siliques with lower seed sets, suggesting the importance of NAD(H) accumulation during the development of pollen. The AtNUDX7 mRNA is expressed ubiquitously in all plant tissues (Ogawa et al., 2005). However, there was no difference in growth or morphology between the control plants and the plants in which AtNUDX7 was overexpressed or disrupted, probably because the altered expression of AtNUDX7 does not lead to a disturbance in the accumulation NAD⁺, which accounts for the majority of the NAD(P)(H) pool (Fig. 5).

To consider that redox equivalents might rapidly cross different compartments, we analyzed effects of the overexpression or disruption of AtNUDX7 on the levels and redox status of primary reductants, AsA and GSH, in plants. However, there was no difference in the levels of AsA, DAsA, GSH, and GSSG in the control, Pro_35S:AtNUDX7, and KO-nudx7 plants under both normal conditions and oxidative stress (data not shown). It is well known that most of the AsA and GSH in green tissues are found in the chloroplasts (Noctor and Foyer, 1998; Meyer and Hell, 2005; Mullineaux and Rausch, 2005; Ishikawa and Shigeoka, 2008). Similarly, 30% to 40% of total cellular NADH is in the chloroplast (estimated concentration, 0.4 mM), and the remaining 45% to 55% and 10% to 15% are in the cytosol (0.65 mM) and the mitochondrion (2.4 mM), respectively (Wigge et al., 1993). Therefore, it is reasonable that the levels and redox states of AsA and GSH are not affected by NADH metabolism as a result of AtNUDX7 in the cytosol. A similar result was observed in mitochondrial complex I-deficient tobacco (Nicotiana tabacum) plants, which respire through alternative respiratory dehydrogenases. These plants showed a 2-fold increase in leaf contents of NAD⁺ and NADH without an appreciable increase in NADP⁺, NADPH, hydrogen peroxide, AsA, or GSH (Dutilleul et al., 2003, 2005).

AtNUDX7 Is Indispensable for Defense against Oxidative Stress

Recently, several researchers have demonstrated that AtNUDX7 functions in the immune responses to pathogens, although doubts remain about the direct effect of AtNUDX7 on the responses (Bartsch et al., 2006; Jambunathan and Mahalingam, 2006; Ge et al., 2007; Adams-Phillips et al., 2008). AtNUDX7 is likely associated with defense systems against oxidative stress, since cellular redox status is closely related not only to the response to pathogenic infection but also to various forms of abiotic stress (Foyer and Noctor, 2005). Based on the indispensable roles of NADH in numerous metabolic reactions, the action of AtNUDX7 is thought to be disadvantageous in the cells. Therefore, the expression of AtNUDX7 might be finely tuned in response to oxidative stress. In fact, the expression of AtNUDX7 was temporarily induced in response to various stressful conditions (Fig. 3). Similar expression patterns of AtNUDX7 are observed in the Genevestigator Arabidopsis microarray database (Zimmermann et al., 2004). According to the information from the database, it is notable that AtNUDX7 is highly 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 treatments with elicitor. Furthermore, the Pro_35S:AtNUDX7 plants having high levels of AtNUDX7 expression clearly showed enhanced tolerance to oxidative stress caused by PQ treatment compared with the control plants (Fig. 4). In contrast, the KO-nudx7 plants showed enhanced sensitivity to the stress. These findings suggest that the actions of AtNUDX7 are indispensable for the systems of defense against oxidative stress.

AtNUDX7 Modulates the PAR Reaction in Response to Oxidative Stress

While it has long been thought that the major cellular functions of NAD⁺ and NADH are to modulate cellular energy metabolism in organisms, increasing evidence has suggested that NAD⁺ and NADH also play key roles in cell death and various major cellular functions, such as Ca²⁺ homeostasis and gene expression (Rutter et al., 2001; Berger et al., 2004). In particular, NAD⁺ has been implicated in the control of gene expression through the PAR reaction; NAD⁺ must be continually produced for the normal functioning of PARP. Furthermore, Ca²⁺ appears to be an important cofactor in PARP's hyperactivation (Bentle et al., 2006). It was noteworthy that, in the Pro_35S:AtNUDX7 and KO-nudx7 plants, the amount of poly(ADP-Rib) correlated with the expression level of AtNUDX7 (Fig. 5). NADH and NAD⁺ pass through the nuclear envelope (Zhang et al., 2002). These findings suggest that AtNUDX7 functions in the modulation of the PAR reaction in response to oxidative stress either directly by the metabolism of NADH in the cytosol or indirectly by perturbation of the NADH/NAD⁺ ratio or by subsequent disruption of Ca²⁺ homeostasis. Notably, suppression of the PAR reaction by treatment with 3-AB enhanced the sensitivity to oxidative stress (Fig. 7), suggesting that activation of the PAR 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 expression of genes encoding PARPs and PARGs in Arabidopsis was induced by various stressful conditions (Doucet-Chabeaud et al., 2001; Ogawa et al., 2009). Furthermore, the activation of poly(ADP-Rib) metabolism reflecting the activation of PARPs occurred in Arabidopsis under oxidative stress caused by PQ treatment (Fig. 5; Ogawa et al., 2009). It is well established that hypersynthesis of PAR causes depletion of NAD⁺ and ATP, leading to energy failure and cell necrosis (Los et al., 2002; De Block et al., 2005). Accumulation of NAD⁺ and ATP was observed in Arabidopsis plants in which the PARP activity was reduced by gene silencing (De Block et al., 2005). Under stressful conditions, the activation of the PAR reaction in the control plants caused decreased levels of NAD⁺ and ATP (Fig. 5, B and C). The excess activation of the PAR reaction in the Pro_35S:AtNUDX7 plants resulted in a decrease in ATP level even under normal conditions (Figs. 2 and 5). On the other hand, NAD⁺ and ATP were accumulated at high levels in the KO-nudx7 and 3-AB-treated plants, in which the PAR reaction was suppressed, under normal and stressful conditions (Figs. 5 and 8). These results clearly indicated that activation of the PAR reaction causes depletion of NAD⁺ and ATP in Arabidopsis 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 reported that the ADP-Rib produced from poly(ADP-Rib) is rapidly degraded to AMP and that the production of AMP might be an important pathway to reestablish utilizable energy units. In addition, it has been demonstrated that an important function of ATP is derived from poly(ADP-Rib) for the maintenance of the replication apparatus during DNA repair (Maruta et al., 2007). Furthermore, nicotinamide was released by the degradation of NAD⁺ by PARP and returned to NAD⁺ by the salvage pathway, which was accompanied by ATP consumption (Noctor et al., 2006). By the action of AtNUDX7 as an ADP-Rib pyrophosphohydrolase, ADP-Rib is hydrolyzed to AMP and Rib 5-P (Ogawa et al., 2005). It has been demonstrated that adenylate kinase catalyzes a reversible transphosphorylation reaction 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 to synthesize ATP from ADP and free phosphate (Wu and Ort, 2008; Zhang et al., 2008). These findings and the levels of ADP-Rib, NAD⁺, and ATP in the control, KO-nudx7, and Pro_35S:AtNUDX7 plants (Fig. 5) suggest that, in addition to NAD⁺ supply coupling to the novel salvage pathway as described above, AtNUDX7 functions in the maintenance of NAD⁺ by supplying ATP through nucleotide recycling from free ADP-Rib molecules under the stress. Consequently, similar to the case of AtNUDX2 (Ogawa et al., 2009), it was reasonable that the overexpression of AtNUDX7 confers enhanced tolerance to oxidative stress in Arabidopsis plants.

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

Cellular DNA, RNA, and their precursor nucleotides are at high risk of being oxidized by ROS under oxidative stress (Nakabeppu et al., 2006). PARP detects SSB generated directly or resulting from the processing of damaged bases by the SSBR/BER pathway and activates the repair reaction (Schreiber et al., 2006). The lack of PARP rendered cells significantly sensitive to alkylating agent and -radiation (Trucco et al., 1998). The acute hypersensitivity and the high genomic instability in PARP-null mice to alkylation caused dramatic decreases in DNA strand-break rejoining (de Murcia et al., 1997). It has been demonstrated that PARP interacts with an adaptor protein, XRCC1, which has two interfaces with two important SSBR/BER enzymes, DNA ligase III and DNA polymerase β (Caldecott et al., 1995, 1996; Kubota et al., 1996; Masson et al., 1998). In addition, Rad51 is a key factor in homologous recombination for the repair of double-strand DNA breaks (Lees-Miller and Meek, 2003; Dudás and Chovanec, 2004). It has been shown that the expression of Rad51 is negatively correlated with PARP activity, since an absence of PARP activity increases the amount of SSB converted into double-strand DNA breaks (Schultz et al., 2003). Numerous homologs of E. coli RecA, AtRAD51, AtDMC1, AtRAD51B, AtRAD51C, AtRAD51D, AtXRCC2, and AtXRCC3, were identified in Arabidopsis (Klimyuk and Jones, 1997; Doutriaux et al., 1998). Disruption of Arabidopsis XRCC2 (Atxrcc2) conferred hypersensitivity to the DNA cross-linking agent mitomycin C (Bleuyard and White, 2004). Similarly, Atxrcc3 mutants were hypersensitive to mitomycin C (Bleuyard et al., 2005). These findings clearly indicated that the E. coli RecA-like protein is involved in recombinational repair in Arabidopsis.

The expression levels of AtXRCC1 and -2 paralleled that of AtNUDX7 under both normal conditions and oxidative stress (Fig. 6). On the other hand, an inverse correlation was observed between the levels of AtXRCC3, AtRAD51, AtDMC1, and AtMND1 and AtNUDX7. As in the KO-nudx7 plants, the down- or up-regulation of those genes was observed in the 3-AB-treated plants (Fig. 9). These findings suggest that the expression of those genes encoding factors related to the repair of DNA is either positively or negatively regulated by the PAR reaction in Arabidopsis and that the regulation contributes, in part, to the tolerance to oxidative stress. Therefore, it is likely that AtNUDX7 is involved in the regulation of defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Materials and Plant Growth Conditions

The vectors for the Gateway cloning system, pDONR201 and pGWB2, were obtained from Dr. 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) on Murashige and Skoog (MS) medium under a light intensity of 100 µE m^–2 s^–1.

Generation of Transgenic Plants

Total RNA was 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'-AAAAAGCAGGCTATGGGTACTAGAGCTCAG-3') and attB2-AtNUDX7 (5'-AGAAAGCTGGGTTCAGAGAGAAGCAGAGGC-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 L^–1 hygromycin, and 20 mg L^–1 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 µg each) extracted from the leaves of 4-week-old transformants (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 expression 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. Protein 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 NADH Pyrophosphohydrolase 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 colorimetrically 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 mL^–1 bovine serum albumin, and crude extract (approximately 10.0 µg 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 ammonium molybdate 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 previously (Ogawa et al., 2008). Chemiluminescence was detected using a luminometer (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 µg) 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 x 250 mm) and AG11 guard (4 x 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 µL of water, and analyzed by CE-ESI-MS/MS. CE-ESI-MS/MS analyses were performed on a P/ACE MDQ capillary electrophoresis 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 x 50 µm 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 of –4.5 kV was 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 on MS medium containing the agent at 2 to 3 µM for 0 to 7 d under normal light (100 µE m^–2 s ^–1) or for 0 to 12 h under high light (1,600 µE m^–2 s ^–1). 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 µM 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 µg) extracted from Arabidopsis 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 Biosystems) were as follows: AtNUDX7-F (5'-CTTGGGATTCGCCATTGTG-3'), AtNUDX7-R (5'-CATGATCCGCATTGCAGTAGAT-3'), AtDMC1-F (5'-AGCCAGCAGGTGGTCATGTACT-3'), AtDMC1-R (5'-ACTGCGACAATGGTGTTCAAAC-3'), AtNMD1-F (5'-ATTCCCGCCTCCGTGTATAGAT-3'), AtNMD1-R (5'-CGCCTTTGCCTTTCCTGAA-3'), AtRAD51-F (5'-AAACCCAGCACGGACCTTTC-3'), AtRAD51-R (5'-AGCATCCCTAAGCTTCTTTACATCAA-3'), AtRAD51B-F (5'-CCTTCCCGTTCCATATAACATCAG-3'), AtRAD51B-R (5'-TGATTCCTGGACCTTTCAGTTCA-3'), AtRAD51C-F (5'-CAAAGTTTAGTGAAGGCTCGTTTCA-3'), AtRAD51C-R (5'-CTCGGTTGGTGCACGAATG-3'), AtRAD51D-F (5'-CTGGAGACAAAGAGACGGACTCA-3'), AtRAD51D-R (5'-GAGGAAGGTCCGACAAGTTCTGT-3'), AtXRCC1-F (5'-GGATGAAGGACCAACCGAAGA-3'), AtXRCC1-R (5'-AACTTGGCTCGGCGTGTTC-3'), AtXRCC2-F (5'-CGCCACTTCACCGTGTACCT-3'), AtXRCC2-R (5'-CGTAGATGCGCCGGTGAT-3'), AtXRCC3-F (5'-TGCAGAAGGATCCGGAGATG-3'), AtXRCC3-R (5'-GGCTGAATTGATCCTCTCGAACT-3'), Actin2-F (5'-GGCAAGTCATCACGATTGG-3'), and Actin2-R (5'-CAGCTTCCATTCCCACAAAC-3'). Quantitative RT-PCR was performed with an Applied Biosystems 7300 Real Time PCR System (Applied Biosystems) using the SYBR Premix ExTaq (Takara). Actin2 mRNA was 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).

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

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

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shigeru Shigeoka (shigeoka{at}nara.kindai.ac.jp).

^[OA] Open Access articles can be viewed online without a subscription.

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

^* Corresponding author; e-mail shigeoka{at}nara.kindai.ac.jp.

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