Nitric Oxide Contributes to Cadmium Toxicity in Arabidopsis by Promoting Cadmium Accumulation in Roots and by Up-Regulating Genes Related to Iron

Uptake1[W]
Angelique Besson-Bard, Antoine Gravot, Pierre Richaud, Pascaline Auroy, Celine Duc, Frederic Gaymard, Ludivine Taconnat, Jean-Pierre Renou, Alain Pugin, and David Wendehenne* UMR INRA 1088/CNRS 5184/Universite de Bourgogne, Plante-Microbe-Environnement, 21065 Dijon cedex, France (A.B.-B., A.P., D.W.); UMR 118 Amelioration des Plantes et Biotechnologies Vegetales, INRA/ Agrocampus Rennes/Universite Rennes 1, 35653 Le Rheu cedex, France (A.G.); Laboratoire de Bioenergetique et Biotechnologie des Bacteries et Microalgues, SBVME, IBEB, DSV, CEA, CNRS, Universite Aix Marseille, 13108 Saint Paul lez Durance, France (P.R., P.A.); Laboratoire de Biochimie et Physiologie Moleculaire des Plantes, UMR 5004 Agro-M/CNRS/INRA/UMII, 34060 Montpellier cedex 1, France (C.D., F.G.); and Unite de Recherche en Genomique Vegetale, UMR 8114 CNRS/INRA/Universite dEvry-Val dEssonne, 91057 Evry, France (L.T., J.-P.R.)

Nitric oxide (NO) functions as a cell-signaling molecule in plants. In particular, a role for NO in the regulation of iron homeostasis and in the plant response to toxic metals has been proposed. Here, we investigated the synthesis and the role of NO in plants exposed to cadmium (Cd2+), a nonessential and toxic metal. We demonstrate that Cd2+ induces NO synthesis in roots and leaves of Arabidopsis (Arabidopsis thaliana) seedlings. This production, which is sensitive to NO synthase inhibitors, does not involve nitrate reductase and AtNOA1 but requires IRT1, encoding a major plasma membrane transporter for iron but also Cd2+. By analyzing the incidence of NO scavenging or inhibition of its synthesis during Cd2+ treatment, we demonstrated that NO contributes to Cd2+triggered inhibition of root growth. To understand the mechanisms underlying this process, a microarray analysis was performed in order to identify NO-modulated root genes up- and down-regulated during Cd2+ treatment. Forty-three genes were identied encoding proteins related to iron homeostasis, proteolysis, nitrogen assimilation/metabolism, and root growth. These genes include IRT1. Investigation of the metal and ion contents in Cd2+-treated roots in which NO synthesis was impaired indicates that IRT1 up-regulation by NO was consistently correlated to NOs ability to promote Cd2+ accumulation in roots. This analysis also highlights that NO is responsible for Cd2+-induced inhibition of root Ca2+ accumulation. Taken together, our results suggest that NO contributes to Cd2+ toxicity by favoring Cd2+ versus Ca2+ uptake and by initiating a cellular pathway resembling those activated upon iron deprivation.

Nitric oxide (NO) is a hydrophobic gaseous molecule and a diffusible free radical. In animal cells, NO is catalyzed from L-Arg by the heme-containing enzymes nitric oxide synthases (NOS). It serves as a signaling molecule and, when produced by the immune system, acts as a cytotoxic agent against invading pathogens or
` This work was supported by the Commissariat a lEnergie Atomique, the Toxicologie Nucleaire Environnementale program, ` Ministere de lEducation Nationale de la Recherche et de la Technologie (fellowship no. 192752005), the Agence Nationale de la Recherche (grant no. BLAN072_184783), and the LOreal France UNESCO-Academie des Sciences (Pour les Femmes et la Science Program, National France Award 2007). * Corresponding author; e-mail wendehen@dijon.inra.fr. The author responsible for distribution of materials integral to the ndings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David Wendehenne (wendehen@dijon.inra.fr). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.133348 1302
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tumor cells (Schmidt and Walter, 1994). The pleiotropic effects of NO in biological systems are related to its ability to react with molecular oxygen, superoxide anion, metal cations, and thiols. In particular, NO modulates the activity of a broad range of proteins by binding to critical Cys residues and to heme or ironsulfur centers (Stamler et al., 1992). Depending on its surrounding microenvironment, notably the presence of other free radicals, the activity and localization of NOS isoforms, and its overall level, NO was shown to exhibit antioxidant or prooxidant effects (Melino et al., 2000). When produced at higher concentrations under pathophysiological contexts, NO is believed to display prooxidant effects and to promote cytotoxic actions contributing, for instance, to neurodegeneration or inammation (Radi, 2004). Almost a decade has passed since the realization of the importance of NO in plant biology (Delledonne et al., 1998; Durner et al., 1998). NO was shown to participate in a wide spectrum of physiological pro-

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Nitric Oxide Contributes to Cadmium-Induced Toxicity

cesses, including germination, root growth, gravitropic bending, control of the timing of owering, stomatal closure, and growth regulation of pollen tubes (Wilson et al., 2007; Besson-Bard et al., 2008b). Furthermore, NO has also been implicated in the plant adaptive response to biotic and abiotic stresses, notably by acting as a signaling molecule (Gould et al., 2003; Delledonne, 2005). It is becoming apparent that NO mediates its effects through S-nitrosylation of Cys residues of target proteins, modulation of protein kinase activities, and mobilization of free Ca2+ and other second messengers, including cyclic GMP and cyclic ADP-Rib (Lamattina et al., 2003; Lindermayr et al., 2006; Romero-Puertas et al., 2007; Courtois et al., 2008). These processes are supposed to be a part of NO-dependent pathways leading to the activation or repression of various genes (Delledonne, 2005; Grun et al., 2006). Several studies provided insights to how NO is produced in plant cells. Besides nonenzymatic production, a nitrite-dependent reaction involving nitrate reductase (NR) has been discovered. Indeed, NR was shown to catalyze the reduction of nitrite to NO in several physiological situations, such as abscisic acidinduced stomatal closure and hypoxia (Dordas et al., 2004; Bright et al., 2006). Many studies have highlighted the occurrence of a second enzymatic process in which NO is produced from L-Arg by a putative NOS-like enzyme. Accordingly, mammalian NOS inhibitors were shown to efciently suppress the NO production in various contexts, ranging from salt stress to plant attacks by pathogens (Besson-Bard et al., 2008b). Furthermore, NOS activities have been measured in plant tissue extracts as well as in several puried organelles (del Ro et al., 2004; Corpas et al., 2006). Since there is no clear homolog of animal NOS in the plant genomes sequenced so far, it is assumed that the plant enzyme displaying NOS activity might be structurally distinct from mammalian NOS. Several studies have investigated NO production during plant exposure to metals, including aluminum and iron. Exposure of Hibiscus moscheutos roots to aluminum led to an inhibition of NOS activity and consequently of NO production in root apical cells (Tian et al., 2007). Similarly, NO production in the transition zone of Arabidopsis (Arabidopsis thaliana) roots was shown to be completely abolished in re sponse to aluminum treatment (Illes et al., 2006). In contrast, an increased NO production has been detected in Arabidopsis cell suspensions upon iron supply (Arnaud et al., 2006). In this context, NO appears to be a key element in the signal transduction pathway leading to the increase of the iron-storage protein ferritin at both mRNA and protein levels (Murgia et al., 2002; Arnaud et al., 2006). Interestingly, Graziano and Lamattina (2007) provided evidence that NO is also produced in iron-decient conditions in the root epidermis of tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) plants. Their analyses of NO functions upon iron deprivation led to the nding that NO promotes the transcriptional activation of the iron assimilationPlant Physiol. Vol. 149, 2009

related genes FER, LeFRO1, and LeIRT1. FER is believed to be a transcription factor that positively regulates LeFRO1 and LeIRT1 genes encoding a ferric chelate reductase and a ferrous iron transporter, respectively, that function together for iron uptake from the soil (Ling et al., 2002). Therefore, depending on iron availability, plant cells use NO as an intracellular signal to promote iron sequestration or uptake, highlighting a central function for NO in the control of iron homeostasis. Cadmium (Cd2+) is a heavy metal displaying toxic effects in plants. It is taken up by roots and can be translocated into aerial organs, where it preferentially accumulates in trichomes on the leaf surface (Salt et al., 1995). Cd2+ pollution is of major concern, since it hampers plant growth by triggering inhibition of photosynthesis and nitrogen metabolism and by decreasing water and mineral nutrient uptake. Moreover, Cd2+ accumulation in crops compromises their commercial value and presents a potential risk to human health. The possibility that plant exposure to Cd2+ might modulate NO production has been reported, but conicting results have been published regarding the impact of Cd2+ on NO production. Depending on the biological model, either Cd2+-mediated induction (Bartha et al., 2005; Kopyra et al., 2006) or inhibition (Rodrguez-Serrano et al., 2006) of NO production has been reported. In the corresponding studies, treatment of plants with articially generated NO was shown to protect plant tissues against the oxidative damage triggered by Cd2+ by promoting the scavenging of reactive oxygen species (ROS) directly through chemical processes or indirectly via the activation of ROSscavenging enzymes (Kopyra et al., 2006; Noriega et al., 2007). Although informative, these studies did not take into account the possibility that NO might be endogenously produced in response to Cd2+ and, therefore, might exert specic roles in this particular physiological context. In this study, we address the questions of NO synthesis and functions in Arabidopsis plants exposed to Cd2+. We provide evidence that Cd2+ application leads to NO synthesis both in roots and leaves. This NO production, which does not involve NR and AtNOA1 (for Nitric Oxide-Associated1; previously known as AtNOS1), is sensitive to mammalian NOS inhibitors and requires the root iron transporter IRT1 (for IronResponsive Transporter1). Furthermore, we combined transcriptomic, biochemical, pharmacological, and genetic analyses to investigate the functions of NO produced in Cd2+-treated plants. Our results support a model in which NO contributes to the metal-induced root growth inhibition. This process might be related to the ability of NO to favor Cd2+ accumulation in roots by promoting Cd2+ versus Ca2+ uptake and by partly counteracting the Cd2+-induced repression of FIT (for Fe-deciency Induced Transcription Factor), FRO2 (for Ferric Reductase Oxidase2), and IRT1 genes, the Arabidopsis orthologs of the tomato FER, LeFRO1, and LeIRT1 genes.
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RESULTS Cd2+ Induces NO Production in Arabidopsis Roots and Leaves

NO production in Arabidopsis was investigated using 4,5-diaminouorescein diacetate (DAF-2DA), a membrane-permeable derivative of the NO-sensitive uorophore 4,5-diaminouorescein (DAF-2). Reactive nitrogen species derived from NO oxidation nitrosate DAF-2 to yield the highly uorescent DAF-2 triazole (DAF-2T; Kojima et al., 1998). DAF-2 and DAF-2 derivatives have been successfully used to detect NO production both in plants and animals (Kojima et al., 1998; Gould et al., 2003; Corpas et al., 2006). Roots were preincubated with DAF-2DA for 2 h, treated by 200 mM Cd2+ for 7 h with or without pharmacological agents, and observed in phase contrast or in uorescence (Fig. 1). Compared with control

plants (Fig. 1, MO), Cd2+ induced a strong increase of DAF-2T uorescence in roots of Arabidopsis plantlets (Fig. 1, AC). This increase of uorescence was specically due to NO, as it was almost completely suppressed by the NO scavenger 2-(4-carboxyphenyl)4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yl-oxy3-oxide (cPTIO; Fig. 1, DF). The Cd2+-induced NO production in roots was also signicantly reduced by the mammalian NOS inhibitors Nv-nitro-L-Arg-methyl ester (L-NAME; Fig. 1, GI) and S,S#-(1,3-phenylenebis(1,3-ethanediyl))bis-isothiourea (PBITU; Fig. 1, JL), suggesting the possible occurrence of a NOS-like enzyme in this process. Interestingly, an increase of DAF-2T uorescence was also observed in leaves of plantlets treated by 50 mM Cd2+ for 96 h at the root level, suggesting that Cd2+ transported from roots to leaves could trigger NO synthesis in leaves (Fig. 2). The ability of Cd2+ to

Figure 1. NO production in Arabidopsis roots exposed to Cd2+. Roots of 3-weekold Arabidopsis seedlings grown on plates were loaded with 20 mM DAF-2DA for 2 h and then treated with 200 mM Cd2+ for 7 h in the absence or presence of 1 mM cPTIO, 5 mM L-NAME, or 1 mM PBITU. Roots were observed in phase contrast (A, D, G, J, and M), in uorescence (B, E, H, K, and N), or both (C, F, I, L, and O). A to C, Cd2+-treated roots. D to F, Cd2+ + cPTIO-treated roots. G to I, Cd2+ + L-NAME-treated roots. J to L, Cd2+ + PBITU-treated roots. M to O, Control (water-treated) plants. Results are from one of four representative experiments.

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produce NO in roots, the measured Cd2+-induced NO synthesis was identical to that observed in the wild type (data not shown). Taken together, our data indicate that Cd2+ induces NO synthesis both in roots and leaves and that neither AtNOA1 nor NR catalyzes this production.
NO Contributes to Cd2+-Induced Reduction of Root Growth

To investigate the physiological function of NO in the plant response to Cd2+, Arabidopsis seedlings
Figure 2. NO production in leaves of Arabidopsis plants treated by Cd2+ at the root level. Roots of 2-week-old Arabidopsis plants grown on plates were treated with 50 mM Cd2+ for 96 h. Leaves were detached, inltrated with 20 mM DAF-2DA, and then observed in phase contrast (A) or in uorescence (B). Results are from one of three representative experiments.

induce a NO production in leaves was veried by monitoring the time course for NO accumulation in Cd2+-treated Arabidopsis leaf discs. As expected, 200 mM Cd2+ triggered an increase of DAF-2T uorescence after 3 h of treatment and remained constant thereafter (Fig. 3A). A slight NO production also occurred in control leaf discs. This background production might be related to a constitutive NO production and/or the wounding occurring during leaf disc preparation. As observed for roots, the Cd2+-induced NO production in leaf discs was almost totally prevented by 1 mM cPTIO (84% inhibition) and reduced by the mammalian NOS inhibitors L-NAME (5 mM) and PBITU (500 mM), with 74% and 52% inhibition, respectively (Fig. 3B). To further characterize the source of NO during Cd2+ treatment, similar experiments were performed in the atnoa1 mutant impaired in the expression of the AtNOA1 gene encoding an enzyme initially thought to catalyze NO synthesis from L-Arg (Crawford et al., 2006; Moreau et al., 2008). The mutant atnoa1 was reported to be impaired in NO synthesis or accumulation in response to abscisic acid or lipopolysaccharide treatments (Guo et al., 2003; Zeidler et al., 2004). Our data indicate that the Cd2+-induced NO synthesis was not suppressed in atnoa1 (Fig. 3B). To complete this analysis, we next investigated the putative contribution of NR as an enzymatic source for NO production. NR has been found to catalyze, both in vitro and in vivo, the NAD(P)H-dependent reduction of nitrite to NO (Yamasaki and Sakihama, 2000; Kaiser and Huber, 2001; Morot-Gaudry-Talarmain et al., 2002). Therefore, we tested NO production in response to Cd2+ in leaf discs of the nia1 nia2 NR-null mutant impaired in both NR1 and NR2 gene activities. As shown in Figure 3B, nia1 nia2 leaf discs produced a signicant level of NO in response to 200 mM Cd2+, in the same manner as did wild-type leaf discs. Similarly, when these mutants were examined for their ability to
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Figure 3. NO production and origin in Cd2+-treated Arabidopsis leaf discs. A, NO production in Cd2+-treated Arabidopsis leaf discs. Leaf discs from 7-week-old Arabidopsis plants grown on soil were vacuum inltrated with 20 mM DAF-2DA, maintained for 1 h in the dark, and then incubated with 200 mM Cd2+. NO production was followed by measuring DAF-2T uorescence. Each value represents the mean 6 SD of 30 measurements (10 replicates per experiment performed three times). B, Origin of Cd2+-induced NO production. Leaf discs from 7-week-old Arabidopsis wild-type (WT), atnoa1, and nia1 nia2 plants were treated by 200 mM Cd2+ in the absence or presence of 1 mM cPTIO, 5 mM L-NAME, or 500 mM PBITU. NO production measured after 20 h of treatment is expressed as a percentage of the maximal response measured in wild-type leaf discs in response to Cd2+. The experimental procedure was the same as described for A. Each value represents the mean 6 SD of 30 measurements (10 replicates per experiment performed three times). 1305

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were grown for 3 weeks on plates containing 30 mM Cd2+ with or without 0.5 mM L-NAME or 250 mM cPTIO. As reported previously (Thomine et al., 2000), Cd2+ triggered an inhibition of root growth (Fig. 4). In the presence of L-NAME or cPTIO, the roots were 33% 6 8% and 36% 6 3.5% longer that those of plants grown with Cd2+ alone, respectively, indicating that NO contributes to Cd2+-induced root growth inhibition. The effects of L-NAME and cPTIO were signicant according to the ANOVA using the LSD test. Interestingly, a reduction of root growth by 21% 6 1.5% or 16.5% 6 2.5% was also observed when seedlings were grown on plates containing L-NAME or cPTIO alone, respectively. This observation might be related to NO involvement in root growth as reported by several studies (Besson-Bard et al., 2008a). Finally, it should be specied that the countering effects of cPTIO and 2+ L-NAME on Cd -induced root growth inhibition were more pronounced when reduced concentrations of Cd2+ were used. Indeed, when plants were treated for 4 weeks by 10 mM Cd2+ and 1 mM L-NAME or by 10 mM Cd2+ and 250 mM cPTIO, the root growth inhibition was reverted by 85% 6 47% and 159% 6 97%, respectively (Supplemental Fig. S1).
Identication of NO-Regulated Root Genes during Cd2+ Treatment

To investigate the mechanisms underlying the effects of NO in roots of Cd2+-treated plants, a genomescale expression proling of Arabidopsis roots was performed using CATMA arrays covering 22,089 nuclear genes (Crowe et al., 2003; Hilson et al., 2004) in order to identify root genes regulated through a NOdependent process. For this purpose, plants were grown hydroponically and then were treated for 24 h

with 30 mM Cd2+ and/or L-NAME. Changes in root gene expression were investigated after the treatment by comparing control plants with L-NAME-, with Cd2+-, and with Cd2+ + L-NAME-treated plants. An additional condition evaluating the effects of 15 mM Cd2+ on gene expression was also performed. We selected as NO target genes all genes whose modulation by 30 mM Cd2+ was modied or completely abolished by L-NAME. Technically, it was not possible to test the impact of the NO scavenger because of the high amount of cPTIO powder required for a full assay. The microarray analysis revealed that 783 genes, corresponding to 254 up-regulated and 529 downregulated genes, display signicant differential expression (Bonferroni P value, threshold of 5%) after 24 h of treatment with 30 mM Cd2+ (Supplemental Fig. S2). Most of these genes (54% and 84% of the up- and down-regulated genes, respectively) were also found to be modulated in response to 15 mM Cd2+ (Supplemental Fig. S2). Among the 783 genes, we identied 43 NO-dependent genes. These genes, which are similarly modulated by 15 and 30 mM Cd2+, were gathered into two classes: (1) genes whose differential expression in response to 30 mM Cd2+ was completely suppressed in Cd 2+ + L -NAME-treated plants, L -NAME alone being without effects (Fig. 5); and (2) genes whose down-regulation by 30 mM Cd2+ was drastically amplied in plants cotreated by Cd2+ + L-NAME, the treatment with L-NAME alone leading to a repression, but to a lower extent, of their expression (Fig. 6). Importantly, 34 of the 43 NO-regulated genes have been reported to be differentially expressed in response to Cd2+ treatment (Connolly et al., 2003; Herbette et al., 2006; Weber et al., 2006). Furthermore, six of these genes were previously reported to be modulated by NO donors (Huang et al., 2002; Parani et al., 2004; Badri et al., 2008). These include At2g03760 encoding a putative steroid sulfotransferase, At5g18170 encoding the Glu dehydrogenase1 (GDH1), At1g08090 encoding the high-afnity nitrate transporter NRT2.1, At1g77760 encoding NR1, and At2g44080 and At3g20340 encoding proteins with unknown function. In addition, orthologs of three of the selected genes, namely IRT1, FRO2, and FIT (Fig. 6), were shown to be modulated by a NO donor in roots of tomato plants (Graziano and Lamattina, 2007). These consistencies with previous studies indicate that our experimental conditions are suitable to identify NO/Cd2+-regulated genes.
Description of the Class I NO-Regulated Genes

Figure 4. NO involvement in Cd2+-triggered root growth inhibition. Arabidopsis seedlings were grown vertically for 3 weeks on plates containing 30 mM Cd2+ and supplemented or not with 250 mM cPTIO or 0.5 mM L-NAME. Primary root length was measured using Optimas version 6.0. The mean 6 SE of four independent experiments is represented. Means with a same letter are not signicantly different at P = 0.05. 1306

This rst class includes 39 genes (27 up-regulated and 12 down-regulated genes; Fig. 5). Three of these genes, At1g56430, At3g56240, and At5g26690, are potentially related to metal homeostasis. At1g56430, also named NAS4 (for Nicotianamine Synthase4), is one of the four NAS genes present in the Arabidopsis genome (Becher et al., 2004). NAS genes encode the enzymes nicotianamine synthases, which catalyze the trimerization of molecules of S-adenosylmethionine to form
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Figure 5. Class I NO-regulated genes. Annotation refers to automatic Arabidopsis annotation according to Arabidopsis Genome Initiative (AGI) number from The Institute for Genomic Research. The Cd 15, Cd 30, Cd 30 + L-NAME, and L-NAME ratio columns indicate CATMA results in a log2 ratio obtained for comparisons between 15 mM Cd2+-, 30 mM Cd2+-, 30 mM Cd2+ + L-NAME-, and L-NAME-treated plants and untreated plants. A statistical cutoff, P , 0.05 after Bonferroni correction (see color code), was used to determine which genes were differentially expressed. A positive ratio indicates that the gene is induced (red), and a negative ratio indicates that the gene is repressed (green).

nicotianamine (NA), a ubiquitous metal chelator (Herbik et al., 1999; Mari et al., 2006). In graminaceous plants (strategy II plants), NA is the precursor of mugineic acid family phytosiderophores and, consequently, plays a key role in iron uptake. In nongraminaceous plants (strategy I plants), NA is thought to have a role in metal long-distance transport and transfer within the cells and might act as a sensor for iron availability (Bereczky et al., 2003; Douchkov et al.,

2005; Le Jean et al., 2005; Mari et al., 2006). At3g56240 (also named CCH for Copper Chaperone) and At5g26690 are two other metal-related genes encoding proteins containing a heavy metal-binding domain. While a role for At5g26690 in metal homeostasis has not been unraveled yet, CCH encodes a 121-amino acid protein showing similarity to Anti-oxidant1, a yeast soluble copper chaperone protein acting in the transport and/ or portioning of copper and protecting cells against

Figure 6. Class II NO-regulated genes. For details, see Figure 5 legend. Plant Physiol. Vol. 149, 2009 1307

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ROS toxicity (Himelblau et al., 1998). CCH may serve a similar role and appears to be involved in copper recycling from senescing leaves to the growing organs (Himelblau et al., 1998; Mira et al., 2001). Three of the class I NO-regulated genes are related to nitrogen assimilation and metabolism, namely NRT2.1 (At1g08090), NR1 (At1g77760), and GDH1 (At5g18170). NRT2.1 expression is up-regulated by Cd2+ through NO, whereas NO mediates Cd2+-induced NR1 and GDH1 repression. Interestingly, both NRT2.1 and GDH1 have been related to root growth. Indeed, NRT2.1 was reported to function as a repressor of lateral root initiation independently of nitrate uptake (Little et al., 2005), and a GDH1-decient mutant was shown to display an impaired root growth phenotype when grown under either intermediate or high inorganic nitrogen conditions (Melo-Oliveira et al., 1996). Thus, the respective up- and down-regulations of NRT2.1 and GDH1 by NO (Fig. 5) correlate with the observation that NO contributes to Cd2+-induced root growth inhibition. Three other genes of the class I NO-regulated genes, At1g79320, At4g05490, and At1g59970, encode proteins related to proteolysis. At1g79320 encodes a putative caspase-like protein, At4g05490 encodes FBL22, displaying a ubiquitin protein ligase activity, and At1g59970 encodes a putative metalloprotease. All of these genes are up-regulated through NO, highlighting the importance of proteolysis in the NO mode of action. The other class I NO-regulated genes encode proteins implicated in a variety of functions, including defense and stress response, cell wall plasticity modulation, detoxication, sulfur metabolism, signal transduction, transcriptional regulation, and metabolism. The two genes showing the highest difference of expression, At5g05340 and At5g39190, encode a putative peroxidase and GER2, a germin-like protein, respectively. Several studies proposed that germin-like proteins might function primarily as superoxide dismutases and/or oxalate oxidases and serve to protect plants from the effects of oxidative stress induced by a range of both biotic and abiotic stresses (Khuri et al., 2001). GER2 was found to be resistant to protease degradation but did not display oxalate oxidase activity (Membre et al., 2000). The details of its function in Arabidopsis are still missing. Finally, four genes encoding proteins with no characterized function or homology to known proteins have also been identied.

2002). It works in concert with the plasma membrane ferric chelate reductase FRO2 (Robinson et al., 1999), which reduces ferric iron in ferrous iron, which in turn is transported through the plasma membrane by IRT1. Colangelo and Guerinot (2004) demonstrated that the transcription factor FIT regulates the level of mRNA accumulation of FRO2 and the levels of mRNA and protein accumulations of IRT1 under iron-decient conditions. Similarly, they showed that At5g03570, which encodes a putative iron transporter, was under the control of FIT. Our observation that L-NAME amplied the Cd2+-induced down-regulation of IRT1, FRO2, FIT, and At5g03570 and also repressed their expression when used alone indicates that NO, produced in response to Cd2+ but also constitutively expressed, up-regulates the expression of these genes. These data, together with the identication of NAS4, At5g26690, and CCH as NO target genes, highlight a functional link between NO and metal homeostasis.
Verication of the Microarray Results

Plants were grown hydroponically and treated for 24 h with 30 mM Cd2+ and/or L-NAME as described above. Three independent assays were performed. Then, for each assay, we selected seven representative genes (GER2, NAS4, NR1, GDH1, IRT1, FRO2, and FIT) belonging to both classes of NO-regulated genes for quantitative real-time PCR analysis to verify the microarray data. The results presented in Supplemental Figure S3 showed good agreement with the microarray data. Indeed, L-NAME efciently suppressed the Cd2+-induced expression of NAS4 and GER2 and the Cd2+-repressed expression of NR1 and GDH1. Furthermore, down-regulation of IRT1, FRO2, and FIT by 30 mM Cd2+ was amplied in plants cotreated by Cd2+ and L-NAME, with L-NAME alone leading to a repression, but to a lower extent, of their expression.
Inhibition of NO Synthesis Leads to a Reduction of Cd2+ Accumulation in Roots

Description of the Class II NO-Regulated Genes

Remarkably, the four genes forming this class, IRT1, FRO2, FIT, and At5g03570, encode proteins related to iron uptake (Fig. 6). IRT1, the founding member of the large ZIP (for Zinc-regulated transporter 1/IRT1-like Protein) family, is a divalent plasma membrane cation transporter essential to the uptake of ferrous iron from the soil in strategy I plants. IRT1 is also a major transporter of heavy metals, including Cd2+ (Vert et al.,
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As discussed above, IRT1 is a major plasma membrane transporter of Cd2+ (Vert et al., 2002). Therefore, we investigated whether the up-regulation of the corresponding gene by NO in Cd2+-treated plants might be related to Cd2+ accumulation in roots. Such a process could partly explain NOs involvement in Cd2+-induced inhibition of root growth. Therefore, we measured metal and ion contents of plantlets grown hydroponically and incubated for 24 h with 30 mM Cd2+ in the presence or in the absence of L-NAME. After this treatment, the metal and ion contents of roots but also of leaves were measured using inductively coupled plasma-optical emission spectrometry (ICP-OES). ICP-OES analysis did not show any signicant difference in sulfur, zinc, copper, magnesium, sodium, potassium, and manganese contents between Cd2+and Cd2+ + L-NAME-treated plants (Table I). However,
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Table I. Effect of L-NAME on the ion and metal contents of roots and leaves of Cd 2+-treated plants Roots of 5-week-old hydroponically grown Arabidopsis plants were exposed to 30 mM Cd2+ for 24 h in the absence or presence of 4 mM L-NAME. Metal and ion contents of roots and leaves were measured by ICP-OES just after treatment. Bold type represents the major differences in the ion or metal content between Cd2+-treated and Cd2+ + L-NAME-treated plants. Bold, italic type represents the impact of Cd2+ treatment on iron content. Each value represents the mean 6 SD of nine measurements (three replicates per experiment performed three times). Values shown are mg g21 dry weight.
Ion Roots Control
L-NAME

Leaves Cd
2+

Cd

2+

+ L-NAME

Control

L-NAME

Cd2+

Cd2+ + L-NAME

Cadmium 0.01 6 0.00 0.02 6 Calcium 37.80 6 8.12 34.44 6 Iron 23.00 2.30 24.77 6 Sulfur 48.74 6 4.86 45.89 6 Zinc 5.96 6 1.21 5.76 6 Copper 0.09 6 0.02 0.07 6 Magnesium 11.33 6 2.84 12.38 6 Sodium 14.55 6 1.82 14.26 6 Potassium 160.87 6 21.21 142.69 6 Manganese 0.25 6 0.07 0.22 6

0.02 4.11 0.21 2.65 0.46 4.89 26.31 4.92 37.49 9.40 3.46 15.80 2.21 17.79 6 1.76 2.42 47.39 6 1.87 46.59 6 4.13 0.67 3.37 6 0.29 3.20 6 0.48 0.01 0.10 6 0.02 0.11 6 0.02 1.46 11.27 6 1.28 11.22 6 2.32 1.18 14.65 6 0.46 14.40 6 0.93 13.00 140.21 6 16.32 134.67 6 19.99 0.04 0.21 6 0.04 0.22 6 0.04

0.01 44.98 0.60 39.17 0.95 0.03 28.56 1.77 131.53 0.31

6 6 6 6 6 6 6 6 6 6

0.02 0.00 6 4.06 47.30 6 0.30 0.55 6 5.12 36.81 6 0.09 1.04 6 0.01 0.03 6 1.75 29.94 6 0.41 1.96 6 0.51 134.40 6 0.05 0.36 6

0.00 1.56 6 2.55 43.67 6 0.10 0.47 6 4.54 37.42 6 0.07 0.73 6 0.00 0.02 6 2.29 27.40 6 0.51 1.76 6 5.23 132.75 6 0.02 0.30 6

0.27 1.42 6 4.12 41.06 6 0.14 0.43 6 3.23 37.54 6 0.05 0.77 6 0.00 0.02 6 1.80 27.08 6 0.38 1.76 6 1.03 133.64 6 0.02 0.29 6

0.23 4.19 0.07 3.93 0.05 0.00 1.54 0.13 4.75 0.03

quantication of the Cd2+ content in the same plants revealed that in the presence of L-NAME, the root Cd2+ content was reduced by 35%. These differences were not observed in leaves in which Cd2+ content was similar in both conditions. These data indicate that NO might be required for Cd2+ accumulation in roots, a function tting well with its ability to up-regulate IRT1, FRO2, and FIT (Fig. 6) and to promote the deleterious effects of Cd2+ on root growth. Interestingly, in Cd2+-treated plants, the root concentration of Ca2+ ion was also reduced by almost 30% (Table I). This reduction was completely suppressed when plants were cotreated with Cd2+ and L-NAME. Here, too, these differences were not observed in leaves. These data suggest that, together with Cd2+, Ca2+ transport and/or accumulation in roots are regulated through a NO-dependent process. Because L-NAME reduced the Cd2+ concentration in roots, it might be hypothesized that the modication in the transcription prole of the selected genes in the presence of L-NAME (Figs. 5 and 6) could be related to the lower Cd2+ concentration rather than to NO itself. However, we found that in plants treated with 15 mM Cd2+, the root Cd2+ concentration was lower than in roots cotreated with 30 mM Cd2+ + L-NAME (data not shown). These data indicate that the effect of L-NAME on the selected genes was related to the inhibition of NO synthesis and not to the reduced Cd2+ content.
NO Production in Roots Is Related to Cd2+-Induced Iron Deciency

grown hydroponically and incubated for 24 h with 30 mM Cd2+. As expected, the ICP-OES analysis shown in Table I revealed that Cd2+ treatment reduced the root iron concentration by 31%. We next measured the production of NO in response to Cd2+ treatment in roots of the irt1 mutant impaired in the expression of IRT1 (Fig. 7). As a positive control, roots of irt1 were exposed to sorbitol, an inducer of hyperosmotic stress. Sorbitol was previously shown to trigger NO synthesis both in plant cell suspensions and tissues (Gould et al., 2003; Lamotte et al., 2006). Remarkably, whereas sorbitol promoted a strong NO synthesis (Fig. 7G), the Cd2+-induced NO production was completely suppressed in irt1 (Fig. 7F). Finally, we investigated the impact of a cotreatment of Arabidopsis wild-type roots with an equimolar concentration of Cd2+ and Fe2+. In this condition, compared with Figure 7B, in which the effect of Cd2+ alone was measured, a reduced NO production was observed (Fig. 7D). It should be mentioned that iron itself also triggered a weak production of NO (Fig. 7C), as reported previously in Arabidopsis cell suspensions (Arnaud et al., 2006).
DISCUSSION

The fact that Cd2+ competes for iron uptake through IRT1 (Vert et al., 2002) combined with the nding that iron deciency results in NO production as reported in tomato roots (Graziano and Lamattina, 2007) led us to assume that the Cd2+-induced NO production may be related to iron deciency. To support this hypothesis, we rst analyzed the iron content of roots of plantlets
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The past several years have revealed much about the role of NO in plants. The emerging picture is that NO is a ubiquitous signal involved in major physiological processes such as root growth, owering, and response to biotic and abiotic stresses (Besson-Bard et al., 2008b). Several studies have also dealt with the antioxidative role of NO in response to heavy metal stresses, including Cd2+ (Kopyra and Gwozdz, 2003; Wang and Yang, 2005; Yu et al., 2005). However, the appreciation of NO function in this context relied on the use of articially released NO. The possibility that NO might be endogenously produced and, therefore, play specic roles in response to heavy metals has been poorly investigated. In this study, we analyzed
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Figure 7. NO production in irt1 and wild-type roots exposed to Cd2+ or to Cd2+ and/or Fe2+, respectively. Roots of 3-week-old Arabidopsis irt1 and wild-type seedlings grown on plates were loaded with 20 mM DAF-2DA for 2 h and then treated with 200 mM Cd2+ for 7 h in the presence or absence of 200 mM Fe2+. Sorbitol (250 mM) was used as a positive control for NO production. A, Control wild-type (WT) roots. B, Cd2+-treated wild-type roots. C, Fe2+-treated wild-type roots. D, Cd2+ + Fe2+-treated wild-type roots. E, Control irt1 roots. F, Cd2+-treated irt1 roots. G, Sorbitol-treated irt1 roots. Results are from one of four representative experiments.

the function of NO produced in Arabidopsis plants exposed to Cd2+. We provided evidence that Cd2+ induced a signicant NO synthesis that contributes to the metal toxicity by favoring inhibition of root growth. Based on a microarray analysis, we identied several Cd2+-dependent genes regulated through a NOdependent pathway and highlight a key role for NO in the control of genes related to iron/Cd2+ transports.
Cd2+ Induces NO Synthesis in Arabidopsis Roots and Leaves

We demonstrated that Cd2+ treatment of roots triggered an increase of DAF-2T uorescence in roots within several hours. The increase of uorescence was completely abolished by the NO scavenger cPTIO, thus suggesting that the enhancement of uorescence intensity was indeed caused by NO. This nding is in line with previous reports showing that Cd2+ mediates NO production in pea (Pisum sativum), Brassica juncea, and wheat (Triticum aestivum) roots and in Glycine max cell suspensions (Bartha et al., 2005; Kopyra et al., 2006; Groppa et al., 2008). Furthermore, it parallels the situation encountered in animals, in which Cd2+ was shown to induce NO production in various cultured cell lines (Hassoun and Stohs, 1996; Ramirez et al., 1999). However, our data are contradictory with those of Rodrguez-Serrano et al. (2006), who reported that 28-d-old pea plants grown for 14 d with 50 mM Cd2+ displayed a reduction in the NO content in lateral and principal roots. It should be mentioned that in that study, NO production was investigated only after 14 d of Cd2+ treatment and not during the early stages of Cd2+ exposure, as we did in our work. Therefore, regarding the difference in monitoring NO production, it appears difcult to strictly compare both stud ies, and we assume that the data of Rodrguez-Serrano
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et al. (2006) do not exclude the possibility that NO might be produced within hours in pea roots. Interestingly, application of Cd2+ at the root level also triggered an increase of DAF-2T uorescence in the leaves. Because Cd2+ was previously shown to be translocated from roots to leaves (Salt et al., 1995), it is tempting to speculate that the increase of uorescence observed in leaves could be related to an effect of the translocated Cd2+. We indirectly addressed this question by showing that Cd2+ induced a continuous NO production in Arabidopsis leaf discs. In this condition, NO production was mainly detected in the stomata and the trichomes on the leaf surface (data not shown), a cell type in which Cd2+ accumulates preferentially (Salt et al., 1995). This observation corroborates our previous data demonstrating that stomata and trichomes represent major sites for NO production in tobacco (Nicotiana tabacum) leaves exposed to several abiotic stresses, including hyperosmotic, heat, and salinity stresses (Gould et al., 2003). Two distinct enzymatic pathways for the generation of NO in plants have been reported: a nitrate/nitritedependent pathway involving NR and a L-Arg-dependent pathway (Besson-Bard et al., 2008b). In that study, the mammalian NOS inhibitors L-NAME and PBITU suppressed Cd2+-induced NO production both in roots and leaf discs. The ability of these compounds to reduce NO production has been reported in many studies (Delledonne, 2005) and suggests that the Cd2+-induced NO production might be catalyzed through a L-Argdependent pathway rather than a nitrate/nitrite-dependent route. Accordingly, NO production was not suppressed in the double mutant nia1 nia2, thus conrming that NR is not a source for NO in this particular physiological context. Similarly, the metal-triggered NO synthesis was still observed in the atnoa1 T-DNA insertional knockout mutant, invalidating the hypothesis
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Nitric Oxide Contributes to Cadmium-Induced Toxicity

that AtNOA1 might contribute to Cd2+-dependent NO production. Therefore, the enzyme catalyzing NO in response to Cd2+ remains unidentied. Several experimental arguments suggest that the Cd2+-induced NO production is, at least partly, related to iron deciency. First, Cd2+ treatment reduced by 31% the iron content in roots; second, IRT1 expression was required for NO synthesis; third, a cotreatment of roots with an equimolar concentration of Cd2+ and Fe2+ led to a reduced NO production. Therefore, based on these ndings, it would make sense that a competition between Cd2+ and iron for uptake through IRT1 leads to a reduced iron inux perceived by root cells as a signal leading to NO production. This possibility is further supported by the study of Graziano and Lamattina (2007) reporting that iron deciency results in NO production in tomato roots.
NO Contributes to Cd2+ Sensitivity of Root Growth

micromolar concentrations of the metal into the culture medium. Then, Cd2+ accumulated linearly in the leaves for a further 6 to 8 h before reaching a gradual saturation. Based on these data, we assume that in our study Cd2+ might rapidly reach a saturated concentration in the leaves before 24 h of Cd2+ treatment. Accordingly, we found that the Cd2+ content in the leaf tissues measured after 96 h of Cd2+ treatment was similar to that measured at 24 h (data not shown). In this scenario, the fact that L-NAME cotreatment affects specically the concentration of Cd2+ in roots suggests that the mechanism underlying this effect does not occur in the early stages of Cd2+ treatment but rather once the leaf Cd2+ content has reached its maximum.
NO Contributes to Root Cd2+ Accumulation

As reported in many studies (Thomine et al., 2000), Cd2+ strongly impaired the growth of Arabidopsis seedlings. This effect was signicantly reverted when the Cd2+-treated seedlings were cultured on a medium supplemented with the NO scavenger cPTIO or L-NAME. Considering that both compounds suppressed Cd2+induced NO synthesis in roots, this indicates a contribution of NO to Cd2+ sensitivity of root growth. A similar observation was recently reported in wheat roots (Groppa et al., 2008). Although speculative, at least two mechanisms might explain this effect of NO. First, this effect might be associated with NOs ability to modulate genes whose expression has been shown to be related to root growth, including NRT2.1 and GDH1. Second, because Cd2+ content in roots of plants grown hydroponically was reduced by 35% in the presence of L-NAME, NOs contribution to Cd2+-triggered root growth inhibition might be linked to its putative involvement in Cd2+ accumulation. This assumption is difcult to reconcile with the fact that, on the contrary, NO was shown to promote root growth, notably by acting downstream of auxin (Correa-Aragunde et al., 2004). Accordingly, we found that seedlings grown in the presence of 2+ L-NAME or cPTIO without Cd displayed a reduced root growth. Thus, it might be hypothesized that, depending on the biological context, NO might exert an opposite effect on root growth. The deleterious effects versus the benecial effects of NO is a common observation that is principally related to the dynamic of its synthesis, that is, the level of produced NO but also the subcellular site of its production and the rate of its diffusion (Melino et al., 2000; Radi, 2004). In contrast to the situation observed in roots, the level of Cd2+ in leaves was not affected by L-NAME cotreatment. This observation should be discussed in the light of the dynamic of Cd2+ transport within plants. Salt et al. (1995) reported that in B. juncea plants grown in a hydroponic system, Cd2+ started to accumulate within 4 to 6 h in the leaves after addition of
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Several studies provided strong evidence that the iron transporter IRT1 is partly responsible for Cd2+ inux into root cells (Clemens, 2006). Supporting this nding, we observed that the Cd2+-mediated NO synthesis was completely suppressed in the irt1 mutant. Data from our microarray analysis indicate that Cd2+ caused a strong reduction of IRT1 but also FRO2 and FIT transcript levels. A similar decline of IRT1 RNA levels, as well as a coordinated down-regulation of both IRT1 and FRO2 genes, was previously reported in plants grown in the presence of Cd2+ (Connolly et al., 2002, 2003). It has been postulated that repression of these genes might avoid the accumulation of metal to toxic levels (Connolly et al., 2002, 2003). Remarkably, in our study, down-regulation of IRT1, FRO2, and FIT genes was exacerbated when plants were cotreated by Cd2+ and L-NAME. This amplied down-regulation corroborated a reduction of the root Cd2+ content, suggesting that NO favors Cd2+ accumulation in roots by up-regulating IRT1, FRO2, and FIT. The observation that plants treated with L-NAME without Cd2+ also displayed reduced transcript levels of these genes further supports a role for NO in their positive regulation. This assumption is also in good agreement with the demonstration that NO produced in tomato roots in response to iron-decient conditions plays a key role in the enhanced accumulation of the mRNA encoding FER, LeFRO1, and LeIRT1, the to-

Figure 8. Hypothetical scheme for NO functions in Arabidopsis roots exposed to Cd2+. NO favors root Cd2+ uptake and therefore contributes to root growth inhibition by partly preventing the Cd2+-induced repression of the Fe starvation-responsive genes IRT1, FRO2, and FIT and by decreasing root Ca2+ content. 1311

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mato orthologs of FIT, FRO2, and IRT1 (Graziano and Lamattina, 2007). The effect of Cd2+ and L-NAME on Ca2+ concentration is also particularly relevant. Indeed, we found that Cd2+ led to a decrease of the root Ca2+ content by 30%. One possible explanation is that both Cd2+ and Ca2+ might compete to enter root cells through plasma membrane Ca2+-permeable channels. This hypothesis is supported by the nding that root plasma membrane Ca2+-permeable channels in wheat have been described as Cd2+ permeant (White, 2000). Furthermore, the wheat Low-Afnity Cation Transporter1, which may function in Ca2+ inux, was shown to mediate the uptake of Cd2+ when expressed in yeast (Clemens et al., 1998). Such Cd2+ permeability of Ca2+permeable channels was also reported in Arabidopsis guard cells and in tobacco cell suspensions (PerfusBarbeoch et al., 2002; Garnier et al., 2006). An alternative explanation might be that Cd2+ also promotes the activation of Ca2+ transporters extruding Ca2+ out of root cells, such as plasma membrane Ca2+-ATPases. Whatever the mechanisms involved, the nding that application of L-NAME completely reverted the inhibitory effect of Cd2+ on Ca2+ accumulation in roots suggests a key role of NO in these processes. The existence of tight interactions between NO and Ca2+ uxes was recently demonstrated. Indeed, NO was shown to promote both activation and/or inhibition of Ca2+ inuxes in plant cells exposed to biotic or abiotic stresses, highlighting its involvement in the control of Ca2+ homeostasis (Besson-Bard et al., 2008b). Mechanistically, pharmacological studies indicate that NO affects the activity of Ca2+-permeable channels, but the possibility that it might also promote the activation of Ca2+ transporters, as reported in animal cells, has been recently discussed (Courtois et al., 2008). Therefore, although the nature of the link between Cd2+ and Ca2+ is unresolved at present, we assume that NO might favor Cd2+ versus Ca2+ uptake and/or Ca2+ extrusion partly by modulating the activity of Ca2+-permeable channels and/or Ca2+ transporters. In summary, this work provides, to our knowledge, the rst mechanistic description of NO putative functions in plants exposed to Cd2+. NO displays a harmful effect by contributing to root growth inhibition. We suggest that this effect may be due to NOs ability to increase Cd2+ accumulation in roots by favoring Cd2+ versus Ca2+ uptake and by initiating a cellular pathway resembling those activated upon iron deprivation via the up-regulation of FIT, FRO2, and IRT1 (Fig. 8). The ongoing analysis of the role of the other NO target genes modulated in response to Cd2+ should open new windows in the understanding of NO function in metal toxicity.

University of California), the NR double mutant nia1 nia2 (a gift from C. Meyer, INRA), and irt1. For growth on plates, seeds were surface sterilized by immersion in a 5% (w/v) calcium hypochlorite/50% (v/v) ethanol solution for 20 min and rinsed three times with distilled sterile water before transferring to square plastic petri plates containing the following medium: 5 mM Ca(NO3)2, 5 mM KNO3, 0.57 mM KH2PO4, 0.97 mM MgSO4, 50 mM H3BO3, 0.05 mM CoCl2, 0.05 mM CuSO4, 15 mM ZnSO4, 2.5 mM KI, 50 mM MnSO4, 3 mM Na2MoO4, 50 mM FeEDTA, and 8 g L21 agar. Plates were placed in the dark at 4C for 3 d and then placed in a climate-controlled growth room for 21 d with the following settings: 16 h of light (350 mE s21 light intensity), 21C, 70% relative humidity, in a vertical orientation so that the roots grew down along the surface of the agar. For growth on soil, plants were cultivated in commercial soil (Jiffy-7; Puteaux) in a climate-controlled growth chamber (KBW 720; Binder) with a 10-h-light (175 mE s21 light intensity)/14-h-dark cycle with the following settings: 20C light/18C dark, 70% relative humidity light/95% dark. For hydroponic culture, seedlings were rst aseptically germinated on solid Murashige and Skoog medium diluted twice. After 2 weeks, the young plantlets were placed on sand for an additional 3-week period in a controlled environment (8 h of light with 300 mE s21 light intensity, 22C, 70% relative humidity). Plants of similar rosette diameters were then transferred to a hydroponic culture using the nutritive solution described by Gravot et al. (2004) for an acclimation of 3 d. L-NAME and CdCl2 treatments were started at 1 and 2 h, respectively, after the start of illumination.

Chemicals
All chemicals were purchased from Sigma-Aldrich except L-NAME and PBITU, which were from ALEXIS Biochemicals. CdSO4, CdCl2, L-NAME, PBITU, and cPTIO were dissolved in water.

Measurement of NO Production
NO accumulation was determined using the uorophore DAF-2DA supplied as a 5 mM solution in dimethyl sulfoxide. Once in the cell, DAF-2DA reacts with intracellular esterases, forming DAF-2. DAF-2 reacts with NOderived species (notably N2O3) resulting from NO oxidation to yield a triazole uorescent derivative (DAF-2T). To assess NO production in roots, roots of Arabidopsis plants grown on plates were loaded with 20 mM DAF-2DA in 50 mM Tris-HCl, pH 7.5, for 2 h in petri dishes in the dark. Then, roots were washed three times with distilled water to wash off excessive uorophore and incubated for 7 h in the dark with CdSO4 in the presence or absence of pharmacological agents or excess iron. DAF-2T uorescence was visualized using a Zeiss Axiovert 200 M inverted uorescence microscope. Digital images were captured with a cool CCD camera controlled with Axiovision software (Zeiss). Twenty-ve roots were analyzed for each condition, and the experiment was repeated four times. A similar procedure was used to check NO production in response to sorbitol. To detect NO production in leaves after exposure of the roots to Cd2+, roots of Arabidopsis plants grown on plates were placed on a CdSO4-supplemented medium. After 96 h of treatment, leaves were harvested, immersed in 20 mM DAF-2DA prepared in 50 mM Tris-HCl, pH 7.5, for 2 h, and washed three times with distilled water. DAF-2T uorescence was visualized using a Leica M2 FP III stereomicroscope. To follow NO production in leaf discs, 7-mm-diameter leaf discs excised from Arabidopsis plants grown on soil were inltrated for 3 min with 20 mM DAF-2DA dissolved in 50 mM Tris-HCl, pH 7.5. Then, leaf discs were incubated for 1 h in the dark, rinsed three times with distilled water, and transferred onto 96-well plates (one disc per well) containing 200 mL of the treatment solution in the dark. NO production was measured using a uorometer (Fluoroskan Ascent uorometer; Labsystems) with 485-nm excitation and 510-nm emission lters. Six leaf discs were used for each treatment. Fluorescence was expressed as relative uorescence units.

Phenotypic Analysis of Cd2+ and NO Effects MATERIALS AND METHODS Plant Material and Growth Conditions
Experiments were performed using Arabidopsis (Arabidopsis thaliana Columbia ecotype) wild type, atnoa1 (formerly atnos1; a gift from N.M. Crawford, Plants were grown on petri plates, as described above. The medium was supplemented with CdSO4 (30 mM) in the presence or absence of cPTIO (250 mM) or L-NAME (0.5 mM) before pouring onto the plates. Root length was measured at the indicated times using Optimas version 6.0. Continuous variables are expressed as means 6 SE. In order to compare values of primary

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root lengths, ANOVA was performed with the statistical program Statgraphics release 5.1 (Manugistic) using the LSD test to detect signicant differences (P , 0.05) between treatments.

Determination of Metal and Ion Concentrations by ICP-OES Experiments

Experiments were carried out using 5-week-old plants transferred to hydroponic culture conditions 3 d before treatments. Cd2+ was applied by feeding the hydroponic solution with 30 mM CdCl2 with or without 4 mM 2+ treatment, leaves and roots from treated and L-NAME. After 24 h of Cd untreated plants were harvested separately. Roots were washed twice with 1 mM EDTA-Na2 solution to remove root-adsorbed Cd2+ and then rinsed with distilled water. The metal and ion contents were determined both in roots and leaves using ICP (ICP-OES Vista MPX).

keeping gene (HK; PDF2: At1g13320) using the comparative threshold cycle method, where Ct represents the threshold cycle for target amplication: DCt = DCtgene of interest 2 DCtHK. The relative transcript levels (RTL) were calculated as follow: RTL = 10 3 22DCt. The following primers were used: AtNAS4-fw (5#-TTATCGGTTTATCACCCTACCG-3#), AtNAS4-rev (5#-TGCGAACTCCTCGATAATGC-3#), NR1-fw (5#-TACGTCGTTGAAATCGCAAA-3#), NR1-rev (5#-GCTGCAACGCAAACTGAAT-3#), GDH1-fw (5#-TGTGGGAGGAAGAGAAGGTG-3#), GDH1-rev (5#-CAGGAGCTGCTAAAGAGATTGC-3#), GER2-fw (5#-ACGTCGGTTTTGTCTCTTCC-3#), GER2-rev (5#-AGGATTGGACCCAAACACAG-3#), IRT1-fw (5#-CGGTTGGACTTCTAAATGC-3#), IRT1-rev (5#-CGATAATCGACATTCCACCG-3#), FRO2-fw (5#-CGTTGCACGAGCGATTCTTG-3#), FRO2rev (5#-GCGACTTGTAGTGCGGCTATG-3#), FIT-fw (5#-GGAGAAGGTGTTTGTCCATCTC-3#), FIT-rev (5#-GGTTAGGCAAGTTTAAGCTCTG-3#), PDF2fw (5#-TAACGTGGCCAAAATGATGC-3#), and PDF2-rev (5#-GTTCTCCACAACCGCTTGGT-3#). Microarray data from this article were deposited at Array Express (http:// www.ebi.ac.uk/arrayexpress/) under accession number E-MEXP-508 and at CATdb (Gagnot et al., 2008; http://urgv.evry.inra.fr/CATdb/; Project RS05-08_NO) according to the Minimum Information About a Microarray Experiment standards.

RNA Isolation
Plants were grown in the same conditions used for ICP-OES experiments. Root tissues were collected 24 h after treatments and immediately frozen in liquid nitrogen before total RNA extraction using Trizol reagent according to the manufacturers instructions (Invitrogen).

Supplemental Data Transcriptome Studies

Microarray analysis was carried out at the Unite de Recherche en Genomique Vegetale using the CATMA array (Crowe et al., 2003; Hilson et al., 2004), containing 24,576 gene-specic tags from Arabidopsis. RNA samples from three independent biological replicates representing three plants per replicate were used. The RNA samples were pooled to minimize the biological variability as described by Peng et al. (2003). The hybridizations were performed with technical replications including uorochrome reversal (dye swap). The reverse transcription of RNA in the presence of Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products), the hybridization of labeled samples to the slides, and the scanning of the slides were performed as described by Lurin et al. (2004). Statistical analysis was performed as described by Lurin et al. (2004). Controls were used for assessing the quality of the hybridizations but were not included in the statistical tests or the graphic representation of the results. For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths of 635 nm (red) and 532 nm (green). No background was subtracted. In the following description, log ratio refers to the differential expression between two conditions. It is either log2 (red/green) or log2 (green/red) according to the experimental design. Array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features. Then, we performed global intensity-dependent normalization using the Loess procedure to correct the dye bias. Finally, for each block, the log ratio median calculated over the values for the entire block was subtracted from each individual log ratio value to correct print tip effects on each metablock. To determine differentially expressed genes, we performed a paired t test on the log ratios, assuming that the variance of the log ratios was the same for all genes. Spots displaying extreme variance (too small or too large) were excluded. The raw P values were adjusted by the Bonferroni method, which controls the family-wise error rate. We considered as being differentially expressed the genes with a familywise error rate of 5%. We use the Bonferroni method (with a type I error equal to 5%) in order to keep a strong control of the false positives in a multiple comparison context (Ge et al., 2003). The following materials are available in the online version of this article. Supplemental Figure S1. NO involvement in 10 mM Cd2+-triggered root growth inhibition. Supplemental Figure S2. Genes signicantly induced or repressed exclusively by 30 mM Cd2+ or by both 15 and 30 mM Cd2+. Supplemental Figure S3. qRT-PCR conrmation of the microarray data.

ACKNOWLEDGMENTS
We are grateful to Annie Buchwalter, Parul Vatsa, and Gerard Vansuyt for helpful discussions and to Andre Bouchot for the microscopical analysis. Received November 27, 2008; accepted January 20, 2009; published January 23, 2009.

LITERATURE CITED
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qRT-PCR Analysis
RNA was isolated from roots with the TRIzol reagent according to the instructions of the manufacturer (Invitrogen). A DNase (Promega) treatment was performed on 5 mg of total RNA to prevent genomic DNA contamination. RNA samples were subsequently used for reverse transcription (MMLV-RT; Promega) with anchored oligo(dT15) (Promega) and 0.4 mM deoxynucleoside triphosphates. The cDNAs were diluted twice with water, and 1 mL of each cDNA sample was assayed by quantitative PCR in a LightCycler (Roche Diagnostics) using FastStart DNA Master PLUS Syber Green I (DeRocher and Bohnert). The efciency of amplication was assessed relative to a sample standard. Expression levels were calculated relative to the appropriate house-

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