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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis^1,[W]

Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, Nara 631–8505, Japan (T.O., H.M., K.I., D.I., N.T., S.S.); 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 CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its K_m value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 µM) than for ADP-glucose (1,235 ± 65 µM). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 µM) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 µM) and NADPH (36.9 ± 3.5 µM). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 µM). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.

A large number of Nudix hydrolases from various organisms such as bacteria, yeast, algae, nematodes, and vertebrates have been identified and characterized (Dunn et al., 1999; McLennan, 2006). For example, the enzymes with pyrophosphohydrolase activity hydrolyze oxidized nucleotides, 8-oxo-7,8-dihydro-2'-(deoxy)guanosine 5'-triphosphate [8-oxo-(d)GTP] from Escherichia coli and human (Maki and Sekiguchi, 1992; Furuichi et al., 1994), NADH from E. coli, Saccharomyces cerevisiae, and human (Frick and Bessman, 1995; Xu et al., 2000; AbdelRaheim et al., 2003), CoA from S. cerevisiae, mice, and Caenorhabditis elegans (Cartwright et al., 2000; Gasmi and McLennan, 2001; AbdelRaheim and McLennan, 2002), ADP-Rib from human, Methanococcus jannaschii, and Synechococcus PCC7002 (Sheikh et al., 1998; Yang et al., 2000; Okuda et al., 2004), and diadenosine polyphosphates (Ap_nA; n = 4–5) from C. elegans and Thermus thermophilus (Abdelghany et al., 2001; Iwai et al., 2004). However, the physiological functions of these subfamilies are still unclear, although some characteristics in vivo, such as subcellular distribution, have been determined (Cartwright et al., 2000; AbdelRaheim et al., 2001, 2003; Gasmi and McLennan, 2001).

In higher plants, surprisingly few attempts have been made at elucidating the characteristics of Nudix hydrolases. Previously, we reported that 24 Nudix hydrolase genes exist in Arabidopsis (Arabidopsis thaliana), and the proteins they encode can be classified into three types by their predicted subcellular localizations: the cytosol (AtNUDX1 to -11), mitochondria (AtNUDX12 to -18), or chloroplast (AtNUDX19 to -24; Ogawa et al., 2005; Yoshimura et al., 2007). Recently, Muñoz et al. (2006) reported that there are seven additional genes encoding Nudix hydrolases in the Arabidopsis genome. Among them, At5g13570 (designated AtDCP2) was characterized as a novel Nudix hydrolase having mRNA-decapping activity (Gunawardana et al., 2008). AtNUDX25 encoded by At1g30110 showed hydrolysis activity toward Ap_nA (Yoshimura et al., 2007). Proteins encoded by At3g10620 (AtNUDX26) and At5g06340 (AtNUDX27) conserved the Nudix motif and were predicted to be located in chloroplasts (Yoshimura et al., 2007). On the other hand, the Nudix motif was hardly conserved in proteins encoded by the remaining three genes (At2g04440, At3g02780, and At5g16440).

We have reported that one of the cytosolic AtNUDXs, AtNUDX1, acts on the hydrolysis of 8-oxo-(d)GTP with high affinity and completely reduces the frequency of spontaneous mutations in the E. coli mutT^– strain (Ogawa et al., 2005). Furthermore, we found that the levels of 8-oxo-guanosine in knockout AtNUDX1 plants significantly increased compared with those in wild-type plants under normal and stress conditions; therefore, AtNUDX1 plays an important role in protection against oxidative DNA and RNA damage in plant cells (Yoshimura et al., 2007). On the other hand, several cytosolic AtNUDXs, AtNUDX2, -6, -7, and -10, had pyrophosphohydrolase activity toward both ADP-Rib and NADH, while AtNUDX11 specifically hydrolyzed CoA (Ogawa et al., 2005). It has been reported that a T-DNA knockout Arabidopsis mutant for AtNUDX7 showed pleiotropic phenotypes, such as reduced plant size, increased levels of reactive oxygen species (ROS) and NADH, microscopic cell death, the constitutive expression of pathogenesis-related genes, and resistance to bacterial pathogens (Jambunathan and Mahalingam, 2006). In addition, Adams-Phillips et al. (2008) have shown that the nudx7-knockout Arabidopsis mutants allow less growth of a virulent pathogen and exhibit a reduced hypersensitive-response phenotype. Bartsch et al. (2006) have demonstrated that AtNUDX7 exerts a negative regulatory effect on ENHANCED DISEASE SUSCEPTIBILITY1 signaling, which controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. Furthermore, Ge et al. (2007) recently reported that AtNUDX7 prefers NADH to ADP-Rib as a physiological substrate and functions in modulation of the defense response to prevent excessive stimulation during both biotic and abiotic stresses.

Among the predicted organelle-type Nudix hydrolases, it has been reported that the predicted mitochondrial AtNUDXs, AtNUDX13 and -14, act on Ap_nA and ADP-sugar, respectively (Muñoz et al., 2006; Olejnik et al., 2007). However, compared with the cytosolic Nudix hydrolases, little is known about the molecular characteristics of predicted organelle-type Nudix hydrolases, although there are many metabolites that are potentially substrates of Nudix hydrolases, such as CoA and its derivatives, NAD(P)H, and FAD, involved in metabolic pathways such as photosynthesis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and β-oxidation in plant organelles. Therefore, to understand the physiological functions of Nudix hydrolases in plants, we analyzed the molecular properties and subcellular distributions of AtNUDX14, -15, -19, -23, -26, and -27 among the predicted organelle-type Nudix hydrolases in Arabidopsis. In addition, we studied the effects of the disruption of organelle-type AtNUDXs on the growth and morphology of Arabidopsis mutants.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Organelle-Type Nudix Hydrolases in Arabidopsis

Predictions of the existence of transit peptide and cleavage sites in the deduced amino acid sequences of all organelle-type AtNUDXs were made with the TargetP prediction program (http://www.cbs.dtu.dk/services/TargetP/; Supplemental Table S1). Consequently, it was predicted that AtNUDX12 to -18 are localized in mitochondria and AtNUDX19 to -24, -26, and -27 are localized in chloroplasts. Similar results were obtained by an analysis using the PSORT prediction program (http://psort.ims.u-tokyo.ac.jp/). Among the predicted organelle-type AtNUDXs, mitochondrial AtNUDX15 and chloroplastic AtNUDX22 contained the conserved motif LLTXR(SA)X₃RX₃GX₃FPGG (designated UPF0035 in the PROSITE database), which is found upstream of the Nudix motif to hydrolyze CoA, like cytosolic AtNUDX11, human NUDT7, mouse NUDT7, yeast Pcd1, and C. elegans Y87G2A.14 (Cartwright et al., 2000; Gasmi and McLennan, 2001; AbdelRaheim and McLennan, 2002; Ogawa et al., 2005; Fig. 1A). The deduced amino acid sequence corresponding to the mature AtNUDX15 shared high homology (73.8%) with that of AtNUDX22.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Consensus motifs of Nudix hydrolases with CoA or NADH pyrophosphohydrolase activities. A, Partial sequence alignment of AtNUDX11, -15, and -22 and other Nudix hydrolases with CoA pyrophosphohydrolase activity from various organisms. Forty-four amino acid sections of the sequence encompassing the UPF0035 motif and the Nudix motif were aligned with the following sequences and accession numbers: ATNUDX11, At5g45940; AtNUDX15, At1g28960; AtNUDX22, At2g33980; yeast Pcd1, NP_013252; human NUDT7, P0C024; mouse NUDT7, Q99P30; C. elegans Y87G2A.14, CAB54476. B, Partial sequence alignment of AtNUDX19 and Nudix hydrolases with NADH pyrophosphohydrolase activity from various organisms. The Nudix motif and SQPWPFPxP motif were aligned with the following sequences and accession numbers: AtNUDX19, At5g20070; human NUDT12, NP_113626; E. coli Orf257, NP_756807; yeast Npy1, P53164.

Complementation of the E. coli mutT Mutation by Mitochondrial and Chloroplastic AtNUDXs

A Nudix hydrolase, E. coli MutT, has oxidized nucleotide pyrophosphohydrolase activity, hydrolyzing all canonical nucleoside triphosphates but with a preference for 8-oxo-(d)GTP, the oxidized form of the free guanine nucleotide, by attacking ROS (Maki and Sekiguchi, 1992; Taddei et al., 1997). These oxidized guanine nucleoside triphosphates would cause both replicational and transcriptional errors, since they pair with adenine and cytosine with almost the same efficiency (Wood et al., 1990; Moriya et al., 1991; Shibutani et al., 1991; Sekiguchi and Tsuzuki, 2002). Therefore, E. coli MutT functions in the prevention of mutagenic consequences. Recently, we demonstrated that one of the cytosolic AtNUDXs, AtNUDX1, acts on the hydrolysis of 8-oxo-(d)GTP and plays an important role in protection against oxidative DNA and RNA damage in plant cells (Ogawa et al., 2005; Yoshimura et al., 2007).

First, to identify an 8-oxo-(d)GTP pyrophosphohydrolase in the predicted organelle-type AtNUDXs, we examined the effect of the expression of these AtNUDXs on the rate of spontaneous mutation toward rifampicin resistance in the E. coli mutT^– strain, CC101T, which was devoid of its own 8-oxo-(d)GTP pyrophosphohydrolase activity (Furuichi et al., 1994). As shown in Supplemental Table S2, the mutation frequency of CC101T cells carrying the cDNAs for each mature AtNUDX protein in the absence of the predicted transit peptide cloned into the pTrc100 expression vector was almost the same as that of the cells transfected with an empty pTrc100. These results suggest that none of the mitochondrial and chloroplastic AtNUDXs contains the MutT-type 8-oxo-(d)GTP pyrophosphohydrolase activity, although it is possible that these AtNUDXs would be inactive following expression in E. coli.

Substrate Specificity of Organelle-Type AtNUDXs

To characterize the recombinant forms of predicted organelle-type AtNUDXs, each recombinant form of the mature AtNUDX proteins in the absence of predicted mitochondrial or chloroplastic transit peptide at the N terminus was produced using E. coli strain BL21 (DE3) pLysS cells and purified from the extract using a HiTrap chelating column. The hydrolytic activities of these enzymes for various types of nucleoside diphosphate derivatives were measured in the presence of 5 mM Mg²⁺ by HPLC (Table I). Among the mitochondrial AtNUDXs, AtNUDX14 hydrolyzed ADP-Rib and ADP-Glc. A reaction product, AMP, was detected in the reaction mixture by HPLC analysis (data not shown), indicating that ADP-Rib and ADP-Glc are hydrolyzed by the enzyme to AMP and Rib 5-P and to AMP and Glc 1-P, respectively. The K_m value for ADP-Rib (13.0 ± 0.7 µM) of AtNUDX14 was approximately 100-fold lower than that for ADP-Glc (1,235 ± 65 µM; Table II). On the other hand, the V_max value for ADP-Glc (30.0 ± 1.40 µmol min^–1 mg^–1) was approximately 2.5-fold higher than that for ADP-Rib (12.5 ± 0.30 µmol min^–1 mg^–1). Mitochondrial AtNUDX15 hydrolyzed CoA similar to cytosolic AtNUDX11, as reported previously (Table I). A reaction product, 3',5'-ADP, was detected (data not shown), indicating that CoAs are hydrolyzed by the enzyme to 3',5'-ADP and 4'-phosphopantetheine. The enzyme showed high affinity for CoA (118.7 ± 3.4 µM) compared with the other CoA pyrophosphohydrolases, such as yeast Pcd1 (Cartwright et al., 2000), mouse NUDT7 (Gasmi and McLennan, 2001), and C. elegans Y87G2A.15 (AbdelRaheim and McLennan, 2002; Table III). No activity toward any other substrate tested here was detected in mitochondrial AtNUDX12, -13, -16, -17, and -18.

Specificity of Mitochondrial AtNUDX15 toward CoA Derivatives

CoA pyrophosphohydrolases, such as S. cerevisiae Pcd1, mouse NUDT7, and C. elegans Y87G2A.14, were active toward various CoA derivatives (Cartwright et al., 2000; Gasmi and McLennan, 2001; AbdelRaheim and McLennan, 2002). Therefore, we examined the substrate specificity for various CoA derivatives of AtNUDXs with CoA pyrophosphohydrolase activity. Mitochondrial AtNUDX15 was highly active toward oxidized CoA the same as yeast Pcd1, while cytosolic AtNUDX11 hydrolyzed malonyl-CoA (Table VI). Interestingly, both enzymes showed high levels of activity toward medium- and long-chain fatty acyl-CoA, including hexanoyl (C_6:0)-CoA, lauroyl (C_12:0)-CoA, myristoyl (C_14:0)-CoA, and palmitoyl (C_16:0)-CoA.

As the Nudix hydrolases require various divalent cations for their activities, we analyzed the effect of several divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, Cu²⁺, and Ca²⁺) on the activities of mitochondrial and chloroplastic AtNUDXs. Mg²⁺ (5 mM) was most effective for the AtNUDX14 activity toward ADP-Rib. The activity in the presence of 5 mM Mn²⁺ was approximately 10% of that in the presence of Mg²⁺. The activities of AtNUDX15 and -19 in the presence of 5 mM Mn²⁺ were 223% and 113%, respectively, of those in the presence of 5 mM Mg²⁺. Zn²⁺ was the most effective divalent ion for AtNUDX23 activity toward FAD. The presence of 5 mM Zn²⁺ resulted in 410% of the activity compared with that with 5 mM Mg²⁺. However, Mn²⁺ and Zn²⁺ concentrations of 5 mM are approximately 1,000-fold higher than typical levels in growing cells (Klaus et al., 2005). The activities of AtNUDX15, -19, and -23 in the presence of 5 µM Mn²⁺ or Zn²⁺ were not detected. Therefore, in most cases, Mg²⁺ is likely to be physically the most relevant divalent cation for all organelle-type AtNUDXs in vivo. The activities of AtNUDX12, -16, -17, -18, -20, -21, -22, and -24 toward any substrates were not observed even under the presence of several divalent cations.

Subcellular Localizations of Predicted Organelle-Type AtNUDXs

Next, we confirmed the subcellular distributions of predicted mitochondrial (AtNUDX14 and -15) and chloroplastic (AtNUDX19, -23, -26, and -27) AtNUDXs, having hydrolysis activities in vitro, in plant cells using their GFP fusion proteins (Fig. 2). The full-length AtNUDX cDNAs, including the transit peptides, were fused in frame with GFP at the C terminus and then transformed into tobacco (Nicotiana tabacum) BY-2 cells or Arabidopsis T87 cultured cells. The fluorescence of the GFP fusion proteins in the transgenic cells was monitored using confocal microscopy. As shown in Figure 2A, the fluorescence of the AtNUDX15-GFP fusion protein was colocalized with mitochondria stained by MitoTraker Orange in the tobacco BY-2 cells. On the other hand, the AtNUDX14-GFP fluorescence was not detected in mitochondria but was colocalized with chlorophyll autofluorescence in the Arabidopsis T87 cells (Fig. 2B), although it was predicted to be distributed in mitochondria based on its deduced amino acid sequence. The fluorescence of the AtNUDX19-GFP fusion protein was detected in both chloroplasts and cytosol, but it was mainly distributed in the chloroplasts. The fluorescence of AtNUDX23, -26, or -27-GFP fusion protein was colocalized with chlorophyll autofluorescence.

View larger version (34K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Subcellular localizations of the predicted organelle-type AtNUDXs. Confocal images of tobacco BY-2 (A) and Arabidopsis T-87 (B) cells expressing the predicted organelle-type AtNUDXs fused with GFP. Plasmids expressing AtNUDXs fused with GFP were transformed to tobacco BY-2 or Arabidopsis T87 cells. Green fluorescence signals (a, b, and g–k) of GFP and red fluorescence signals of mitochondria stained with MitoTracker (c and d) or chlorophyll autofluorescence (l–p) were detected by a laser confocal microscope. Merged images are shown in e, f, and q to u. The detailed procedures are described in "Materials and Methods." Bars = 10 µm.

Semiquantitative reverse transcription (RT)-PCR was performed to determine the tissue-specific expression of predicted organelle-type AtNUDX mRNAs. As shown in Figure 3, almost all of the AtNUDX mRNAs were expressed ubiquitously in the rosette leaves, stems, cauline leaves, inflorescences, and roots. On the other hand, the level of AtNUDX18 mRNA was lowest in the rosette leaves and highest in the inflorescence among the various tissues. The AtNUDX20 mRNA was expressed specifically in the rosette and cauline leaves. The expression of AtNUDX24 mRNA was observed in the inflorescences and rosette and cauline leaves. These results suggest that the expression of AtNUDX18, -20, and -24 mRNAs is regulated in a tissue-specific manner, although their substrate specificities remain unclear. Similar expression patterns of the organelle-type AtNUDXs in the tissues are observed in the GENEVESTIGATOR Arabidopsis microarray database (Zimmermann et al., 2004). According to the detailed information about tissue-specific expression of the organelle-type AtNUDXs from the database, it is notable that AtNUDX12, -13, -15, -16, -21, and -22 are highly expressed in pollen and/or stamen, suggesting an involvement of the proteins in the male reproductive organs.

View larger version (65K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Expression of the organelle-type AtNUDX genes in different plant tissues. Semiquantitative RT-PCR was performed using specific primers for the AtNUDXs and Actin2 genes with total RNA from rosette leaves, stems, cauline leaves, inflorescences, and roots. PCR amplification was performed with 20 to 28 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s, followed by 72°C for 10 min. Aliquots of the products were analyzed on 1% agarose gels. The detailed procedures are described in "Materials and Methods."

To assess the physiological relevance of the individual organelle-type AtNUDXs, we studied effects of the disruption of organelle-type AtNUDXs on the growth and morphology of Arabidopsis mutants. T-DNA insertion Arabidopsis mutants of AtNUDX13 (SALK_058284), -14 (SALK_087382), -15 (SAIL_1255_G04), -19 (SALK_115339 and SALK_135053), -20 (SALK_138802), -21 (SALK_055509), -23 (SAIL_539_H02), -25 (SALK_016093), -26 (SALK_040636), and -27 (SALK_139887) have been registered in the SIGnAL project (http://signal.salk.edu/tabout.html). A complete loss of mRNA expression caused by homozygous T-DNA insertion into the respective AtNUDXs, except for AtNUDX19 and -23, in each mutant was verified by semiquantitative RT-PCR analysis (data not shown). In both T-DNA mutants of AtNUDX19 (SALK_115339 and SALK_135053), the T-DNA insertion locating in the fourth intron resulted in significant suppression (<50%) of the AtNUDX19 expression, indicating an inhibition of pre-mRNA splicing by the insertion. No T-DNA insertion was detected in the genome of the T-DNA mutant of AtNUDX23 (SAIL_539_H02); therefore, the level of AtNUDX23 mRNA was not altered in the mutant. Under long-day conditions (16 h of light [100 µmol m^–2 s^–1], 25°C/8 h of dark, 22°C), no difference between the wild-type plants and the T-DNA mutants (AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27) was observed in growth and morphology throughout the cultivation period (data not shown).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Subfamilies of Organelle-Type AtNUDXs

The substrates for Nudix hydrolases include several compounds that are potentially toxic, cell signaling molecules, regulators of cellular metabolism, or metabolic intermediates. Therefore, it has been suggested that the Nudix hydrolases might be cellular surveillance agents participating in the physiological homeostasis of living organisms. In human cells, the genomic DNA in mitochondria is likely to be more susceptible to ROS-induced oxidative damage because the rate of oxygen metabolism is high; thus, homologs of E. coli MutT (designated MTH1) located both in the cytosol and the mitochondria prevent the misincorporation of oxidized deoxy(ribo)nucleotides such as 8-oxo-(d)GTP and 2-OH-(d)ATP into genomic DNA and mRNA (Kang et al., 1995; Yoshimura et al., 2003). Interestingly, it has been reported that MTH1 functions in the prevention of cell death with mitochondrial degeneration by hydrolyzing oxidized nucleotides (Ichikawa et al., 2008). In plant cells, there are three types of genomic DNA distributed in the nucleus, mitochondria, and chloroplasts. Recently, we reported that AtNUDX1, one of the cytosolic AtNUDXs, acts on the hydrolysis of 8-oxo-(d)GTP and plays an important role in protection against oxidative DNA and RNA damage in plant cells (Ogawa et al., 2005; Yoshimura et al., 2007). On the other hand, none of the mitochondrial or chloroplastic AtNUDXs could suppress the rate of spontaneous mutation caused by the deficiency of MutT in E. coli (Supplemental Table S2) and had activity toward 8-oxo-(d)GTP (Table I). These results suggest that there are no 8-oxo-(d)GTP pyrophosphohydrolases in the mitochondria and chloroplasts of Arabidopsis and that cytosolic AtNUDX1 is the sole enzyme preventing oxidative DNA and RNA damage in the plant cells, although there is a possibility that the organelle-type AtNUDXs are expressed as inactive forms in E. coli CC101T strain.

An analysis of the enzymatic properties of organelle-type AtNUDXs revealed that they could be divided into five subfamilies: ADP-sugar, CoA, NAD(P)H, FAD, and Ap_nA pyrophosphohydrolases. Interestingly, AtNUDX mRNAs encoding the enzymes with pyrophosphohydrolase activities toward a variety of nucleoside diphosphate derivatives detected here were expressed ubiquitously in all plant tissues (Fig. 3); therefore, these AtNUDXs could be considered to play "housecleaning" roles, that is, to clean the cell of potentially deleterious endogenous metabolites and/or to regulate the accumulation of intermediates in diverse biochemical pathways in each organelle. Each possible role based on enzymatic properties analyzed here is discussed below.

ADP-Sugar Pyrophosphohydrolase

Previously, Moreno-Bruna et al. (2001) reported a bacterial Nudix hydrolase, designated ADP-sugar pyrophosphohydrolase (ASPP), which hydrolyzes ADP-sugars, such as ADP-Rib, ADP-Man, and ADP-Glc. Since ADP-Glc is a precursor for glycogen, ASPP is thought to control the intracellular level of a molecule linked to the glycogen biosynthetic process in E. coli. In plants, the hydrolytic activity of ADP-Glc was widely distributed and was inversely correlated with the accumulation of starch accumulation (Rodriguez-López et al., 2000). Recently, Muñoz et al. (2006) showed that AtNUDX14 is not a mitochondrial protein. Here, we demonstrated that AtNUDX14 was distributed in the chloroplasts (Fig. 2). Furthermore, we showed that AtNUDX14 has activities toward ADP-Rib and ADP-Glc; however, the K_m and k_cat/K_m values for ADP-Rib are approximately 100-fold lower and 40-fold higher, respectively, than those for ADP-Glc (Table II). However, AtNUDX14 (AtASPP)-overexpressing Arabidopsis plants exhibited a large reduction in the levels of ADP-Glc and starch, indicating that AtNUDX14 functions in the regulation of intracellular ADP-Glc levels linked to starch biosynthesis (Muñoz et al., 2006). Since the intracellular concentration of ADP-Rib is very low (20–40 µM; Ge et al., 2007), AtNUDX14 seems to prefer the hydrolysis of ADP-Glc to that of ADP-Rib in vivo.

CoA Pyrophosphohydrolase

CoA is an essential carbonyl-activating cofactor utilized in the biosynthesis and catabolism of both primary and secondary metabolites in bacteria, plants, and animals (Tilton et al., 2006). In plants, CoA is crucial for the proper functioning of fatty acid biosynthesis in chloroplasts, the TCA cycle in mitochondria, β-oxidation in glyoxysome and peroxisome, and sterol biosynthesis and amino acid metabolism in cytosol. To date, Nudix hydrolases active toward CoA and its derivatives have been identified in mice, human, and C. elegans, and all of them have the conserved motif UPF0035, found adjacent to the Nudix motif on the N-terminal side (Gasmi and McLennan, 2001; AbdelRaheim and McLennan, 2002). The Nudix hydrolases having CoA pyrophosphohydrolase activity in animals and C. elegans were localized in peroxisomes, suggesting a conserved peroxisomal function for the enzymes. Among the predicted organelle-type Nudix hydrolases in Arabidopsis, the UPF0035 motif was found in AtNUDX15 and -22 (Fig. 1). As expected from the deduced amino acid sequence, AtNUDX15 showed CoA pyrophosphohydrolase activity, as did cytosolic AtNUDX11 (Tables I and III; Ogawa et al., 2005), and was distributed in the mitochondria (Fig. 2A). On the other hand, the predicted chloroplastic AtNUDX22 could not hydrolyze CoA, suggesting the possibility of expression of an inactive form in E. coli. Therefore, it is likely that eukaryotic posttranslational modifications of the protein are required for the activation of AtNUDX22. It is notable that mitochondrial AtNUDX15 and cytosolic AtNUDX11 hydrolyzed CoA derivatives, including not only malonyl-CoA and succinyl-CoA, which are metabolites of fatty acid biosynthesis in chloroplasts and of the TCA cycle in mitochondria, but also oxidized CoA, which is a potentially toxic or nonfunctional form of CoA (Table VI). Furthermore, both AtNUDX15 and -11 showed strong activities toward medium- and long-chain fatty acyl-CoA. The hydrolysis activity toward a wide range of CoA derivatives has been observed in RP2p, a Nudix hydrolase, in mouse kidney peroxisome, although its substantial role is still unclear (Ofman et al., 2006). On the other hand, AtNUDX15 and -11 could not effectively hydrolyze isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of Val and odd- and branched-chain fatty acids (Table VI; Lucas et al., 2007). These results suggest that AtNUDX15 and -11 function in the regulation of oxidized CoA and acyl-CoA levels in mitochondria and cytosol, respectively, and, therefore, are closely associated with the maintenance of the cellular CoA level and CoA-relating metabolism such as the TCA cycle.

NAD(P)H Pyrophosphohydrolase

Nudix hydrolases having NADH pyrophosphohydrolase activity in S. cerevisiae and human showed peroxisomal localization, suggesting that the enzymes function to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors (Xu et al., 2000; AbdelRaheim et al., 2003). Unlike these enzymes, AtNUDX19 was primarily localized in chloroplasts (Fig. 2B) and acted on NADH and NADPH (Table I). The SQPWPFPxS motif is found immediately downstream of the Nudix motif in AtNUDX19 as well as other Nudix hydrolases with NADH pyrophosphohydrolase activity from E. coli (Frick and Bessman, 1995), S. cerevisiae (Xu et al., 2000), and human (AbdelRaheim et al., 2003; Fig. 1B). The K_m and k_cat/K_m values for NADPH were approximately 100-fold lower and 10-fold higher than those for NADH (Table IV), suggesting that AtNUDX19 preferred the hydrolysis of NADPH to that of NADH as a physiological substrate in vivo. The importance of NADH and NADPH as electron carriers in all organisms has long been known, and their multiple functions in energy metabolism, signaling pathways, and detoxification reactions imply that the regulation of the NAD(H)/NADP(H) balance is critical for cell survival (Zeigler, 2000). In plant chloroplasts, NADPH, produced from NADP⁺ in the photosynthetic electron transport chain, provides important reducing energy in chlorophyll synthesis and the Calvin cycle. In spinach (Spinacia oleracea) and sugar beet (Beta vulgaris), 40% of total cellular NADPH is in the chloroplasts, this value reaching 60% to 85% under light irradiation (Hunt et al., 2004). Furthermore, the chloroplast, a major source of ROS, has evolved various antioxidative enzymes that utilize NADPH either directly or indirectly (Mittler, 2002). However, so far, it has remained unclear exactly how the NADPH that pools in different organelles, cells, and tissues is (re)synthesized, degraded, and maintained. By the AtNUDX19 reaction, NADPH and NADH were hydrolyzed to NMNH and 2',5'-ADP and to NMNH and AMP, respectively. It has been reported that human nicotinamide mononucleotide adenyltransferase (NMNAT1) is able to catalyze the synthesis of both NAD⁺ from NMN and NADH from NMNH (Emanuelli et al., 1992). The synthesis of NADPH is achieved by the reduction of NADP⁺ produced by NAD kinase or the phosphorylation of NADH by NADH kinase directly (Chai et al., 2005). Recently, a nicotinamide mononucleotide adenyltransferase (AtNMNAT) was identified in Arabidopsis and characterized (Hashida et al., 2007). Therefore, it is likely that chloroplastic AtNUDX19 plays an important role in modulation of the NADH and/or NADPH pools through the hydrolysis of NAD(P)H to NMNH in chloroplasts.

FAD Pyrophosphohydrolase

The flavin nucleotides, FAD and FMN, participate in numerous vital processes, such as mitochondrial electron transport, photosynthesis, fatty acid oxidation, and the metabolism of vitamins B₆ and B₁₂ and folate in all living organisms. Here, we showed that chloroplastic AtNUDX23 had FAD pyrophosphohydrolase activity, by which FAD was hydrolyzed to FMN and AMP (Tables I and II). Nudix hydrolases having FAD pyrophosphohydrolase activity have been identified in bacteria and bacteriophage (Xu et al., 2002; Tirrell et al., 2006). Recently, the characteristics of a bifunctional enzyme (AtFMN/FHy) with activities of riboflavin kinase and FAD synthetase in Arabidopsis were reported (Sandval and Roje, 2005). However, little is known about the enzymes responsible for the turnover and membrane transport of riboflavin and flavin nucleotides, despite the crucial roles of these nucleotides in metabolism in plants. Three of the plant enzymes involved in the biosynthesis of the riboflavin precursor [lumazine synthase, bifunctional GTP cyclohydrolase II/3,4-dihydrozy-2-butane 4-phosphate synthase, and 2,5-diamino-6-ribosylamino-4(3H)-pyrimidine 5-phosphate deaminase] have been characterized from Arabidopsis (Jordan et al., 1999; Herz et al., 2000; Fischer et al., 2004). Interestingly, the deduced amino acid sequences of all of these enzymes contain chloroplast transit peptides. Therefore, it is suggested that if riboflavin is synthesized only in plastids, mitochondria and the cytosol must either import flavin nucleotides or import riboflavin to synthesize flavin nucleotides (Sandval and Roje, 2005). This hypothesis raises the possibility that the FAD pyrophosphohydrolase, AtNUDX23, functions in the regulation of the ratio of FMN and FAD in whole plant cells.

Ap_nA Pyrophosphohydrolase

Ap_nA is a ubiquitous family of nucleotides in the submicromolar to low micromolar range in which two nucleotide moieties are linked 5'-5' through a polyphosphate chain containing from two to seven phosphoryl groups; Ap₄A is the most widely studied (Fisher et al., 2006). In prokaryotes, Ap₄A has been found to have a number of physiological roles, including the modulation of chaperone and heat shock protein activity and the control of timing of cell division (McLennan, 2006). Furthermore, Ap₄A has been implicated in the control of DNA replication and repair, regulation of ATP-sensitive K⁺ channels, initiation of apoptosis, modulation of Fhit tumor suppressor protein activity in conjunction with Ap₃A, and activation of gene expression (Feussner et al., 1996). Notably, McLennan (2000) reported that if allowed to accumulate, Ap₄A could interfere with a number of ATP-dependent reactions. Therefore, it is likely that the signaling via Ap_nA molecules acts both extracellularly and intracellularly, suggesting that subcellular concentrations of Ap_nA should be finely controlled. The Nudix Ap₄A hydrolase was mainly distributed in the nucleus of tomato (Solanum lycopersicum; Hause et al., 1997). On the other hand, E. coli YgdP and the C. elegans NUDT2 ortholog, Ndx-4, appeared to be ribosome associated (Li et al., 2004; Butland et al., 2005). Recently, an asymmetrical Ap₄A hydrolase was isolated from L. angustifolius and characterized as to the functions of key amino acid residues by site-directed mutagenesis (Maksel et al., 2001). It has been reported that AtNUDX13 had activities specific for long-chain Ap_nA and was localized in mitochondria (Olejnik et al., 2007). Therefore, it is suggested that AtNUDX13 could be involved in the turnover of ATP and ADP in mitochondria, since Ap_nA, which are much more stable than ATP, could serve as a storage medium for the latter compound in mitochondria. Here, we showed that AtNUDX26 and -27 act on Ap_nA derivatives (Tables I and V) and are distributed in chloroplasts (Fig. 2). Therefore, the metabolism of Ap_nA may be of functional importance to several biological reactions in chloroplasts of Arabidopsis.

AtNUDXs with No Activity

The activities of predicted mitochondrial AtNUDX12, -13, -16, -17, and -18 and chloroplastic AtNUDX20, -21, and -24 were not detected with any of the substrates tested here. It has been demonstrated that some proteins with the Nudix motif act on nonnucleotide substrates such as diphosphoinositol polyphosphates (Safrany et al., 1998, 1999), 5-phosphoribosyl 1-pyrophosphate (Fisher et al., 2002), and thiamine pyrophosphate (Lawhorn et al., 2004). Therefore, it seems likely that AtNUDXs with no activity toward any substrate tested here would act on nonnucleotide substrates or be inactive following expression in E. coli and/or purification by affinity chromatography. In addition, it is possible that the predicted cleavage site to remove the transit peptide is not correct for the production of mature enzyme.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
A large number of Nudix hydrolases have been isolated from various organisms, including bacteria and animals, although the physiological role of each is still unclear. Here, we characterized the molecular properties of AtNUDX14, -15, -19, -23, -26, and -27 among the predicted organelle-type Nudix hydrolases in Arabidopsis. These results indicate that there are many Nudix hydrolases varying in substrate specificity in Arabidopsis and strongly suggest that these Nudix hydrolases play diverse roles in regulating a wide range of physiological processes. However, the T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions (data not shown). Accordingly, it is possible that the functions of AtNUDXs are either not essential for development under normal conditions or redundant in Arabidopsis plants. To clarify in more detail the physiological relevance of each organelle-type Nudix hydrolase, we are progressing toward the analysis of intracellular concentrations of important metabolites involved in numerous vital processes such as photosynthesis and the TCA cycle in the T-DNA mutants under various growth conditions.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Materials and Plant Growth Conditions

Arabidopsis (Arabidopsis thaliana ecotype Columbia) plants were grown on basic Murashige and Skoog medium in petri dishes containing 3% (w/v) Suc for 2 weeks and then grown on soil at 25°C under long-day conditions (16 h of light at 100 µE m^–2 s^–1/8 h of dark).

The Escherichia coli strain CC101 and the mutT-deficient strain CC101T were gifts from Prof. H. Maki (Nara Institute of Science and Technology). The plasmid pTrc100 for the complementary assay was obtained from Prof. Y. Nakabeppu (Kyushu University). The 8-oxo-(d)GTP was purchased from TriLink Biotechnologies. Restriction enzymes and modifying enzymes were purchased from TaKaRa. All other materials and enzymes were of analytical grade and obtained from commercial sources. The Arabidopsis Genome Initiative codes for predicted organelle-type Nudix hydrolases in Arabidopsis are shown in Supplemental Table S1.

Complementation Assay of the E. coli mutT Mutation

Total RNA was isolated from leaves of 4-week-old wild-type plants (1.0 g fresh weight). First-strand cDNA was synthesized using ReverTra Ace reverse transcriptase (Toyobo) with an oligo(dT) primer. cDNA fragments encoding each mature AtNUDX protein except for predicted mitochondrial or chloroplastic transit peptides at the N terminus (Supplemental Table S1) were amplified by PCR using the specific primer sets (Supplemental Table S3). The amplified DNA fragment was ligated into pT7 Blue T vectors (Novagen). DNA sequencing was performed using the dideoxy chain terminator method with an automatic DNA sequencer (ABI PRISM 310; Applied Biosystems). The resulting construct was digested with each restriction enzyme and was ligated into the expression vector pTrc100, in which each AtNUDX cDNA was oriented in-frame with the ATG initiation codon immediately upstream of its cloning site.

The complementation assay was carried out according to the method described by Ogawa et al. (2005). The empty pTrc100 or vectors containing each AtNUDX cDNA were introduced into E. coli strain CC101 (wild type) or CC101T (mutT^–). A single transformant was grown in 5 mL of Luria-Bertani (LB) medium containing 100 µg mL^–1 ampicillin at 37°C overnight. The culture solution was diluted 1 x 10⁶-fold, and 100 µL of each culture was grown in 6 mL of LB medium containing 1 mM isopropylthio-β-galactoside for 14 to 16 h. The frequency of mutations leading toward rifampicin resistance was measured by plating aliquots of these cultures on LB medium with or without the antibiotic (100 µg mL^–1).

Expression and Purification of Recombinant AtNUDX Proteins

cDNA fragments encoding each mature AtNUDX except for predicted mitochondrial or chloroplastic transit peptides at the N terminus (Supplemental Table S1) were amplified by PCR from the first-strand cDNA synthesized from total RNA of Arabidopsis using the specific primer sets as shown in Supplemental Table S3. The amplified DNA fragment was ligated into pT7 Blue T vector, digested with the desired restriction enzymes, and recloned into the vector pCold II (TaKaRa). Recombinant forms of each AtNUDX were produced using E. coli strain BL21 (DE3) pLysS cells (Ogawa et al., 2005) and purified from the extract using a HiTrap chelating HP column (GE Healthcare) according to the manufacturer's instructions. Protein content was determined following the method of Bradford (1976). Almost all of the recombinant forms of predicted organelle-type AtNUDXs were produced in E. coli with high efficiency in the soluble fraction. However, the recombinant AtNUDX17, -21, -22, and -24 proteins were detected only in the insoluble fraction, because of the formation of inclusion bodies. Therefore, we recloned these cDNAs into the vector pCold TF, which expresses Trigger Factor chaperone as a soluble tag. As a result, these recombinant proteins were observed in the soluble fraction. All of the recombinant proteins were purified to homogeneity using a HiTrap chelating HP column, as judged with SDS-PAGE (Supplemental Fig. S1). The molecular mass of each recombinant AtNUDX protein agreed with the predicted value, which was calculated from the amino acid sequence of the mature protein plus the hexahistidine tag (Supplemental Table S1; Supplemental Fig. S1).

Enzyme Assay and HPLC Analysis

Hydrolytic activities of the recombinant forms of mitochondrial or chloroplastic AtNUDXs toward various types of nucleoside diphosphate derivatives were assayed according to a method described previously (Ogawa et al., 2005). Sixty microliters of the reaction mixture, containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 5 to 1,000 µM substrate, and 0.2 to 1.0 µg of the purified recombinant protein, was incubated at 37°C for 10 min. The reaction was terminated by adding 10 µL of 100 mM EDTA. In addition, the reaction was carried out under reducing conditions by 1 mM DTT according to Olejnik et al. (2007). The mixture was then analyzed with HPLC using a COSMOSIL C18 column (4.6 x 250 mm; Nacalai Tesque) at a flow rate of 0.6 mL min^–1 for the mobile phase buffer, which contained 73 mM KH₂PO₄, 5 mM tetrabutylammonium dihydrogenphosphate, and 15% to 30% methanol. The substrate and reaction product(s) (as shown in parentheses) were detected according to their UV absorbance, as follows: 8-oxo-(d)GTP (2-oxo-dGMP), 293 nm; dGTP (dGMP), 252 nm; dCTP (dCMP), 271 nm; dTTP (dTMP), 264 nm; ADP-Rib (AMP), NADH (AMP), FAD (FMN), CoA (3',5'-ADP), Ap_nA (ATP, ADP, or AMP), UDP-Glc (AMP), and UDP-Gal (UMP), 260 nm.

Subcellular Localization of GFP Fusion Proteins

The vectors for the generation of the GFP fusion proteins were constructed using Gateway cloning technology (Invitrogen). The cDNAs encoding the open reading frame of AtNUDXs were cloned into the donor vector, pDONR201, and then recloned into the destination vector, pGWB5, in which the AtNUDX proteins fused with GFP at their C termini was expressed under the control of the cauliflower mosaic virus 35S promoter. The specific primers with attB1 and attB2 sequences were as follows: attB1-AtNUDX14-cGFP (5'-AAAAAGCAGGCTATTGATGGCTGGCTTTAC-3'), attB2-AtNUDX14-cGFP (5'-AGAAAGCTGGGTAAGAGTTGGGTTTCAGTC-3'), attB1-AtNUDX15-cGFP (5'-AAAAAGCAGGCTCCGCAAATCGGATTCATG-3'), attB2-AtNUDX15-cGFP (5'-AGAAAGCTGGGTAAGGCATACAAGTATGTT-3'), attB1-AtNUDX19-cGFP (5'-AAAAAGCAGGCTTTCCGGTGCGTATAATGC-3'), attB2-AtNUDX19-cGFP (5'-AGAAAGCTGGGTACGGTTGCAGATGGTAAT-3'), attB1-AtNUDX23-cGFP (5'-AAAAAGCAGGCTTTCCGGTGCGTATAATGC-3'), attB2-AtNUDX23-cGFP (5'-AGAAAGCTGGGTACGGTTGCAGATGGTAAT-3'), attB1-AtNUDX26-cGFP (5'-AAAAAGCAGGCTATGGCACTGTACCGACCC-3'), attB2-AtNUDX26-cGFP (5'-AGAAAGCTGGGTACTGGAGATGAGAAGCGA-3'), attB1-AtNUDX27-cGFP (5'-AAAAAGCAGGCTGCGCCATGGCCGTGAAGG-3'), attB2-AtNUDX27-cGFP (5'-AGAAAGCTGGGTAATCCTTAGACGAGTTCA-3'). PCR and in vitro BP and LR recombination reactions were carried out according to the manufacturer's instructions (Invitrogen).

Agrobacterium tumefaciens (strain C58), which was transformed with the constructs obtained by electroporation, was used for the transformation of tobacco BY-2 (Nicotiana tabacum ‘Bright Yellow-2’) and Arabidopsis T87 cells using a modification of the procedure reported by Gu and Verma (1997). The mitochondria were stained with a mitochondria-selective dye, MitoTracker Orange (Invitrogen). The fluorescence was observed with a Radiance 2100 confocal fluorescence microscope (Bio-Rad). Images were processed using LaserSharp2000 software (Carl Zeiss).

Analysis of AtNUDX Expression in Various Tissues

Total RNA was isolated from various tissues (0.5 g fresh weight), rosette leaves, stems, cauline leaves, inflorescences, and roots, of 6-week-old Arabidopsis plants as described previously (Ogawa et al., 2005). First-strand cDNA was synthesized using ReverTra Ace (Toyobo) with an oligo(dT) primer as described above. cDNAs encoding each AtNUDX and Actin2 were semiquantitatively amplified by PCR using the primer sets shown in Supplemental Table S1. Equal loading of each amplified gene sequence was determined with the control Actin2 PCR product.

Identification of Arabidopsis T-DNA Mutants

The Arabidopsis T-DNA mutants of AtNUDX13, -14, -15, -19, -20, -21, -23, -25, -26, and -27 (obtained through the SIGnAL project; http://signal.salk.edu/tabout.html) were selfed to check for segregation and to obtain a purely homozygous line. Genomic DNA extracted from the leaves of the mutants was used for identification of the T-DNA insertion site by genomic PCR analysis. The cDNA pool was prepared from total RNA extracted from the leaves of 2-week-old Arabidopsis plants. The cDNAs encoding AtNUDX and Actin2 were semiquantitatively amplified by PCR using specific primers in a reaction involving 23 to 27 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s, followed by 72°C for 10 min. Aliquots of the products were analyzed on a 2% agarose gel. Equal loading of each amplified gene sequence was determined with the control Actin2 PCR product. The specific primers for the AtNUDX and Actin2 mRNAs are shown in Supplemental Table S3.

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: AtNUDX12, At1g12880; AtNUDX13, At3g26690; AtNUDX14, At4g11980; AtNUDX15, At1g28960; AtNUDX16, At3g12600; AtNUDX17, At2g01670; AtNUDX18, At1g14860; AtNUDX19, At5g20070; AtNUDX20, At5g19460; AtNUDX21, At1g73540; AtNUDX22, At2g33980; AtNUDX23, At2g42070; AtNUDX24, At5g19470; AtNUDX26, At3g10620; AtNUDX27, At5g06340.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Purification of the recombinant AtNUDXs.

Supplemental Table S1. Molecular characteristic of AtNUDXs.

Supplemental Table S2. Antimutator effect of AtNUDXs in E. coli.

Supplemental Table S3. Primer sequences for AtNUDXs.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Hisaji Maki and Kaoru Yoshiyama (Nara Institute of Science and Technology) and Dr. Yusaku Nakabeppu (Kyusyu University) for generously donating the E. coli mutT^– strain and pTrc100 vector and for excellent technical assistance and helpful discussions.

Received August 26, 2008; accepted September 22, 2008; published September 24, 2008.

	FOOTNOTES

 
¹ This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (grant no. 19039032) from MEXT, Japan, by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists (grant no. 18–1015 to T.O.), by Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (grant no. 2005–2010 to S.S.), and by the Academic Frontier Project for Private Universities: Matching Fund Subsidy from MEXT (grant no. 2004–2008 to S.S.).

² These authors contributed equally to the article.

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).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.128413

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

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