JXB Advance Access originally published online on November 1, 2006
Journal of Experimental Botany 2006 57(15):4111-4122; doi:10.1093/jxb/erl184
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RESEARCH PAPER |
Root-to-shoot long-distance circulation of nicotianamine and nicotianamine–nickel chelates in the metal hyperaccumulator Thlaspi caerulescens
1Biochimie et Physiologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (UMR 5004), Institut National de la Recherche Agronomique, Université Montpellier 2, École Nationale Supérieure d'Agronomie, 2 Place Viala, F-34060 Montpellier cedex 2, France
2Laboratoire de Chimie Bio-Inorganique Environnement, Centre National de la Recherche Scientifique (UMR 5034) Hélioparc, 2 avenue du Professeur Angot, F-64053 Pau cedex 09, France
* To whom correspondence should be addressed. E-mail: czernic{at}univ-montp2.fr
Received 25 April 2006; Accepted 5 September 2006
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Abstract |
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Plant metal hyperaccumulator species are widely used as models to unravel the heavy metal tolerance and hyperaccumulation mechanisms. Thlaspi caerulescens is capable of tolerating and hyperaccumulating Zn, Cd, and Ni. A search for factors involved in the cellular tolerance to Ni, based on yeast screens, led to isolation of a cDNA encoding a functional nicotianamine (NA) synthase (NAS). The T. caerulescens genome appears to contain a single copy of the NAS gene named TcNAS whose expression is restricted to the leaves. The analysis of dose–response and time-course Ni treatments have revealed that the exposure to Ni triggers the accumulation of NA in the roots. Because neither TcNAS expression nor NAS activity were detected in the roots, the NA accumulation in roots is most probably the result of its translocation from the leaves. Once in the roots, NA, together with Ni, is subsequently found in the xylem, for redirection to the aerial parts. Using liquid chromatography coupled to inductively coupled plasma or electrospray ionization mass spectrometry, it has been shown that part of the Ni is translocated as a stable Ni–NA complex in the xylem sap. This circulation of NA, Ni, and NA–Ni chelates is absent in the non-tolerant non-hyperaccumulator related species T. arvense. Taken together, the results provide direct physiological and chemical evidence for NA and NA–heavy metal complex translocation in a hyperaccumulator species.
Key words: Circulation, metal chelation, metal hyperaccumulation, nicotianamine, nickel
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Introduction |
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Some plants have evolved the ability to grow under conditions of high metal concentrations in the soil, such conditions being toxic for most plants (Briat and Lebrun, 1999). Various adaptative mechanisms are responsible for such behaviour. One of them is related to metal hyperaccumulation. Metal hyperaccumulator plants extract metals from the soil and translocate them to their shoots where they are concentrated in a range of 100- to 1000-fold more than in non-hyperaccumulating plants (Brooks et al., 1977; Baker and Brooks, 1989). Metal translocation from the roots to the aerial parts through the xylem is therefore a key determinant of the hyperaccumulation phenotype (Briat and Lebrun, 1999; Clemens, 2001; Clemens et al., 2002). At a molecular level, amino and organic acids have been proposed to play major roles in the heavy metal hyperaccumulation or tolerance phenotype (Sharma and Dietz, 2006). Histidine has been suggested to participate in Ni transport in the xylem of Alyssum lesbiacum (Krämer et al., 1996). However histidine does not appear to be involved in hyperaccumulation of Ni in Thlaspi goesingense, and modulation of histidine content has no effect on Ni tolerance (Persans et al., 1999). Citrate has been demonstrated as one of the ligands binding Ni in the latex of Sebertia acuminata, a New Caledonian tree with 20% of its latex dry mass composed of Ni (Jaffré et al., 1976; Schaumlöffel et al., 2003). Despite these scarce examples, no clear mechanisms of metal chelate long-distance trafficking related to metal hyperaccumulation have been described.
Thlaspi caerulescens is an example of a polymetallic hyperaccumulator plant known to overload Zn and Cd. It belongs to the Brassicaceae family and is phylogenetically close to Arabidopsis thaliana. It has become one of the model plants to study Zn and Cd hyperaccumulation (Lasat et al., 2000; Pence et al., 2000; Assunçao et al., 2001, 2003; Bernard et al., 2004). However, the unusual metal-accumulating ability of T. caerulescens is not restricted to Zn and Cd since it is also able to accumulate Ni (Reeves and Brooks, 1983; Schat et al., 2000). It has also been shown that the Ganges ecotype of T. caerulescens is able to resist and to hyperaccumulate Ni, and that the resistance to Ni was at least partly expressed at the cellular level (Marquès et al., 2004). Based on these findings, a cellular screen was developed in the yeast Saccharomyces cerevisiae and a T. caerulescens cDNA encoding nicotianamine synthase (TcNAS) was isolated that enhanced the yeast resistance to Ni toxicity (Vacchina et al., 2003). Furthermore, using analytical methods combining high-performance liquid chromatography (HPLC) and capillary electrophoresis coupled to various mass spectroscopy techniques, the existence of an Ni–nicotianamine (NA) complex in yeast cells has been demonstrated (Vacchina et al., 2003). NA is a non-proteinous amino acid synthesized in all plants by the condensation of three S-adenosyl-methionine molecules through the activity of the enzyme NAS (Higuchi et al., 1994). NA's critical role in metal homeostasis in plants has mainly been worked out for Fe. It has been exemplified by the study of the tomato mutant chloronerva, which is depleted of NA (Pich et al., 1994; Pich and Scholz, 1996). In this mutant, Cu is blocked in the roots and Fe is not remobilized from the vessels to the mesophyl cells in the leaves, inducing Fe deficiency, which in turn provokes a chlorosis particularly visible in young leaves. NA is also a strong chelator of metals other than Fe in vitro, and can bind Cu, Zn, Mn, and Ni with high affinity (Benes et al., 1983; Stephan and Scholz, 1993; Stephan et al., 1996; Vacchina et al., 2003). In the hyperaccumulator Arabidopsis hallieri, AhNAS2 is part of a set of genes highly and constitutively expressed in the roots that could play a role in Zn tolerance and accumulation (Weber et al., 2004). Arabidopsis halleri presents a 2-fold increase of its NA root content probably linked to the constitutive expression of the AhNAS2 gene. Moreover, the heterologous expression of the AhNAS2 cDNA in a zinc-sensitive Schizosaccharomyces pombe strain leads to an increase in zinc tolerance (Weber et al., 2004). Recently it was reported that the overexpression of TcNAS in A. thaliana transgenic plants also confers Ni resistance (Pianelli et al., 2005), strengthening the idea that NA could play a role in metal tolerance and hyperaccumulation.
In this context, it is therefore of primary importance not only to characterize the site(s) of synthesis of NA but also to determine the various plant organs where NA accumulates, independently of where it is synthesized. Indeed, it is known that NA can be found in the xylem and in the phloem saps, revealing a putative role in the long-distance circulation of metals (Pich et al., 1994; Stephan et al., 1994; Pich and Scholz, 1996; Liao et al., 2000). To demonstrate such a role, beside the measurement of NA and metal contents in various parts of the plant, a key element is the identification of NA–metal chelates in the saps. Indeed, on the basis of the biochemical properties of NA and the phenotype of the chloronerva mutant, it has been widely assumed, almost accepted, that NA is a metal chelator in vivo, although this has never been clearly and unambiguously demonstrated. In this work, it was demonstrated that TcNAS is likely to be encoded by a unique gene that has a constitutive expression restricted to leaves. TcNAS expression and NAS activity were never observed in roots, even after high Ni concentration treatment of the plants. However, upon such Ni treatment, an increase in NA abundance was observed in the roots, which correlated with an increase of the metal content in this organ, as well as an increase in the NA and the Ni amounts in the xylem. Taken together, the results indicate a role for NA in the root-to-shoot translocation of Ni in T. caerulescens, and this is reinforced by the identification of NA–Ni chelates in the xylem of plants treated with Ni.
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Materials and methods |
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Plant growth culture conditions and sampling of leaves, roots, and xylem sap
Seeds from T. caerulescens plants were collected from the metal-contaminated site of Les Malines, Saint Laurent le Minier, France (referred as to the MA population in Escarré et al., 2000). For hydroponic cultures, after germination, seedlings were transferred to a 2.5 l vessel containing a nutrient solution: 1.25 mM KNO3, 1.50 mM Ca(NO3)2·4H2O, 0.75 mM MgSO4·7H2O, 0.50 mM KH2PO4, 50 µM H3Bo3, 19 µM MnCl2, 1 µM CuSO4, 10 µM ZnCl2, 0.2 µM MoO4Na2·2H2O, and 50 µM NaFe-EDTA. The nutrient solution was replaced weekly. For Ni treatment, the nutrient solution was supplemented with 10 or 100 µM NiSO4. Growth conditions were 8 h of light, and 20/15 °C day/night temperatures, respectively. Xylem sap was collected by inserting a capillary tube in decapitated plant stems.
Sequence alignment and phylogenetic tree
The sequence of the TcNAS cDNA has been deposited in the EMBL database, accession no. AJ300446
[GenBank]
. The FASTA (Pearson and Lipman, 1988) and the BLAST algorithms (Altschul et al., 1990) were used to search the DNA and protein databases for similarities. The sequence of the TcNAS protein (Q8GU17) was aligned with those of various dicotyledonous plants (A. thaliana AtNAS1, Uniprot accession no. Q9FF79; AtNAS2, Q9FKT9; AtNAS3, O80483
[GenBank]
; AtNAS4, Q9C7X5; Arabidopsis halleri AhNAS1, Q7OII1; and tomato LeNAS1, Q9XGI7) using the ClustalW software (Thompson et al., 1994). To construct the phylogenic tree, the sequences of NAS proteins from monocotyledonous plants were added to the dicotyledonous NAS sequences referred to above: barley HvNAS1 (Q9ZQV9), HvNAS2 (Q9ZQV7), HvNAS3 (Q9ZQV8), HvNAS4 (Q9ZQV6), HvNAS5 (Q9ZQV5), HvNAS6 (Q9ZQV3), HvNAS7 (Q9ZWH8); maize ZmNAS1 (Q8S9C5), ZmNAS2 (Q8LT19), ZmNAS3 (Q8LT22); and rice OsNAS1 (Q9SXQ7), OsNAS2 (Q9FEG8), and OsNAS3 (Q9FXW5). Calculations were performed using the ClustalW Neighbor–Joining method, and the tree was generated with Tree View X (version 0.4.1 © 2003, Roderic DM Page).
Genomic DNA preparation and Southern blot analysis
Genomic DNA was extracted from 1 g of leaves using the cetyltriethylammonium bromide (CTAB) protocol (Ausubel et al., 1999). A 5 µg aliquot of DNA was digested using EcoRI, HindIII or both enzymes. DNA fragments were separated by electrophoresis in a 0.7% agarose gel in 1x TAE buffer and transferred onto a Hybond N+ charged membrane (Amhersham, Saclay, France). Hybridizations were performed at 65 °C according to the manufacturer using the full-length TcNAS cDNA as a probe after 32P labelling (Roche Dignostics, Meylan, France). The 1 kbp DNA ladder (Gibco-BRL, Life Technologies, Grand Island, NY) was used as molecular marker. The full-length TcNAS cDNA was PCR amplified using the NAS5- (5'-GGC TGC AGG AAT CAA TAT CTT A-3') and NAS3- (5'-ATA CTG CAG AAA AGA CAG CAA C-3') specific primers. After hybridization, the membrane was washed twice in 2x SSC, 0.1% (w/v) SDS at room temperature for 15 min and exposed directly for the low stringency condition or further washed twice with 0.1x SSC, 0.1% SDS for 15 min at 65 °C for the high stringency condition before exposure to a Storm PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
RNA preparation and analysis by northern blot
Total RNA was extracted from roots or leaves using TRIzol reagent (Gibco-BRL), and northern blots were carried out as already described (Czernic et al., 1999) using the same TcNAS probe as indicated above. A 25S rRNA probe was used as a reference, and visualization of the blots was achieved using a Storm PhosphorImager (Molecular Dynamics).
Analytical methods for NA and NAS activity measurements
NA extraction and HPLC detection were performed as already described (Le Jean et al., 2005). Measurements of NAS activity were achieved by incubation of protein extracts (Higuchi et al., 1999) with the bona fide substrate S-adenosyl-methionine. NA was detected after ortho-phthaladialdehyde (OPA) derivatization as described (Le Jean et al., 2005).
Ni content measurement
Leaves and roots of individual plants were harvested, rinsed in distilled water or 15 mM EDTA, respectively, and dried at 80°C for 48 h. About 50 mg of dried material was digested with concentrated HNO3 for 30 min at 250 °C and then diluted with ultra-pure water to 1% HNO3. Ni content was measured by atomic absorption spectrometry (Varian SpectAA 220). Ni content in xylem sap was analysed directly by inductively coupled plasma-mass spectrometry (ICP-MS) after 10-fold dilution with 2% HNO3. The ICP-MS instrument was an ELAN 6000 (Perkin-Elmer SCIEX, Thornhill, ON Canada).
NA–Ni chelate analysis by SE-HPLC-ICP-MS and ESI-MS/MS
Xylem sap samples (100 µl) were eluted with 5 mM ammonium acetate buffer pH 6.2 at a flow rate of 0.75 ml min–1 on a Superdex Peptides HR 10/30 column (Pharmacia Biotech, Uppsala, Sweden). The ICP-MS instrument was an ELAN 6000 (Perkin-Elmer SCIEX, Thornhill, ON Canada). The column eluate was introduced into the ICP via a cyclonic nebulizer. The isotopes monitored were 58Ni and 60Ni. The peaks observed correspond to Ni complexes.
To identify the bio-ligands of Ni in the xylem sap, the procedure used was similar to that detailed by Ouerdane et al. (2006) for the determination of metal complexes in plant leaf extracts. In brief, the sample was injected again and the eluting fraction of size exclusion chromatography (SEC) corresponding to the Ni peak previously detected by ICP-MS was collected off-line. It was freeze-dried, dissolved in 300 µl of 5 mM ammonium acetate in methanol, and then separated by a second chromatographic purification step, hydrophilic interaction liquid chromatography (HILIC), using the same conditions and material as described in Ouerdane et al. (2006). After this second step, the unique fraction containing the metal complex (determined by ICP-MS) was collected again, dried, and dissolved in 100 µl of 50% methanol for eletrospray ionization (ESI)-MS/MS analysis. ESI-MS/MS experiments were performed with a QSTAR Pulsar quadrupole time-of-flight (TOF) hybrid tandem mass spectrometer (AB/MDS SCIEX). The solution was introduced into the microion-spray source at a flow rate of 0.4 µl min–1. The ion-spray voltage was 4800 V (positive mode). The TOF mass analyser was calibrated using a polypropylene glycol standard. The Ni and Ni–NA isotopic patterns were detected as follows: given the relative abundance of 58Ni and 60Ni, a complex containing a single Ni atom will display a mass spectrum consisting of two peaks separated by 2 u, M for 58Ni and M+2 for 60Ni with relative intensities of 
70% and 30%, respectively. A complex containing two Ni atoms will display three peaks, M (58Ni+58Ni, relative intensity: 50%), M+2 (58Ni+60Ni, relative intensity: 38%) and M+4 (60Ni+60Ni, relative intensity: 12%). In the product ion scan mode, to achieve complex identification, the collision-induced dissociation (CID)-MS spectra were obtained for at least two different Ni isotopes (e.g. 58Ni and 60Ni) and then compared with the CID-MS spectra of standards of the expected complexes. The collision energy was optimized for the Ni–NA complex at 40 eV.
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Characterization of nicotianamine synthase in Thlaspi caerulescens
As a first step to evaluate the involvement of NA in the behaviour of T. caerulescens against Ni toxicity, the number of genes encoding NAS within this plant genome was assessed. PCR amplification of T. caerulescens genomic DNA using specific primers for TcNAS (see Materials and methods) gave a single DNA fragment of 1138 bp which is the same size as the corresponding cDNA, indicating that the corresponding gene contained no intron (data not shown). To estimate the number of genes in the Ganges ecotype, a Southern blot using the full-length TcNAS cDNA as a probe was performed (Fig. 1A). Using EcoRI that has a single site in the cDNA, two DNA fragments of 1000 and 4000 bp, respectively, were observed (lane 1). Digestion of the genomic DNA with HindIII that does not cut the cDNA allowed the visualization of a single DNA fragment of 7000 bp (lane 2). With the double digestion (HindIII and EcoRI), two close DNA fragments of
1000 bp were observed (lane 3). The same hybridization pattern was observed after washing the membrane under low stringency conditions (data not shown). Taken together, these data indicate that the genome of the T. caerulescens Ganges ecotype appears to contain a single NAS gene, which was named TcNAS.
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The deduced TcNAS protein sequence was compared with other NAS proteins from dicotyledonous plants. High levels of similarities were observed between TcNAS and NAS proteins from various other plants, ranging from 90% for AtNAS3 and AhNAS1 to 72% for AtNAS4 (Fig. 1B). The single amino acid substitution (phenylalanine to serine at position 238), which abolished the enzyme activity in the tomato chloronerva mutant (Ling et al., 1999), is indicated in Fig. 1B by an asterisk below the alignment. This amino acid is located in a highly conserved region (FLYP) present in all the NAS sequences deposited in the databases originating from both dicotyledonous and monocotyledonous plants.
The comparison of the various plant NAS sequences was extended by building up a phylogenic tree including NAS from graminaceous plants. As shown in Fig. 1C, the closest homologues of the TcNAS protein are the AhNAS1 and AtNAS3 proteins from A. halleri and A. thaliana, respectively (Fig. 1C). As observed above for the nucleic acid sequences, the dicotyledonous proteins remained linked together and were separated from those of monocotyledonous plants, suggesting different evolutionary rates and/or selection pressure between the two types of plants.
TcNAS expression and enzyme activity in response to various nickel concentrations
To investigate the involvement of NA in T. caerulescens upon Ni exposure, plants were grown in hydroponic cultures and exposed to 0, 10, or 100 µM NiSO4 for 1 week. Increasing Ni concentrations resulted in an increase of the Ni content in the roots. This was even more markedly observed in the leaves, where the Ni concentration reached 1.7 mg g–1 dry weight (DW) (Fig. 2A), in good agreement with the hyperaccumulating capacity of T. caerulescens towards Ni (Marquès et al., 2004). Such a behaviour was absent in the non-hyperaccumulating plant T. arvense where Ni accumulated and remained in the roots (Fig. 2B).
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In contrast to the distribution of Ni both in roots and in leaves, TcNAS mRNA accumulation was restricted to the leaves (Fig. 2D). Furthermore, it was expressed at the same level, regardless of the Ni treatment. The slight variations in TcNAS mRNA amounts in the leaves were not reproducibly observed. The organ-specific expression of the TcNAS gene was strengthened by the lack of detectable NAS enzyme activity in the roots, whereas in the leaves a constitutive NAS enzyme activity was measured, varying between 420 and 600 pmol min–1 mg–1 of protein. As observed for the amount of mRNA (Fig. 2B), this leaf NAS enzyme activity was not influenced by the Ni treatments.
Reverse-phase HPLC measurement of NA (Fig. 2C) showed a constant and high NA amount in leaves at either 0, 10, or 100 µM Ni in the medium. The increased leaf NA content upon 10 µM Ni exposure was not reproducibly observed, and was correlated neither to the TcNAS expression nor to the NAS enzyme activity. In sharp contrast, NA was not detected in roots under the control condition (R0 in Fig. 2C). However, a concomitant increase in abundance of both NA and Ni was observed in roots upon exposure to 10 or 100 µM Ni (Fig. 2A, C). Beside Ni, a multielemental analysis of the samples was performed, including Fe, showing no variation in all the other metal ion contents (data not shown). This excludes, therefore, any secondary effect of Ni treatment that could have induced metal deficiency through competition for uptake. The non-tolerant non-hyperaccumulator plant T. arvense displayed a quite different behaviour upon Ni treatment. Constitutive high levels of NA were observed in the leaves (Fig. 2B). After exposure to 10 µM Ni, a dose that caused no visible phytotoxicity, Ni accumulated in the roots (Fig. 2B). However, under this condition, NA did not accumulate in the roots and Ni was not translocated to the leaves (Fig. 2B).
Analysis of TcNAS transcript, and Ni and NA abundance in roots and leaves during a time course of nickel treatment
To go further in the characterization of the role of NA in T. caerulescens, plants were grown in hydroponic cultures and exposed to 100 µM NiSO4 for various periods of time. The measurements of Ni in the roots and the leaves were performed during the time-course (3 weeks) of the exposure to Ni treatment (Fig. 3A). In the roots, Ni was already detectable after 6 h exposure and increased moderately during the 3 weeks of treatment, to reach 1 mg g–1 DW. In the leaves, Ni could be detected after 24 h and then its concentration increased at a much higher rate than in the roots. After 72 h, the shoot-to-root Ni ratio was already above 1, which is one of the characteristic features of plant metal hyperaccumulators. Actually, it reflects the extremely high translocation rates of metal ions from the roots to the aerial parts in this type of plant. In this experiment, the Ni accumulation in the leaves after 3 weeks of exposure was almost three times higher than in the roots, reaching 3 mg g–1 DW (Fig. 3A).
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A northern blot experiment from the same plant samples confirmed the complete absence of TcNAS mRNA accumulation in the roots during the overall period of the experiment (Fig. 3B). In the leaves, the mRNA accumulation levels appeared constitutive (Fig. 3B). Indeed, quantification of the amount of TcNAS mRNA relative to the 25S RNA accumulation indicated a <2-fold variation between the various time points of the time course (data not shown).
For NA quantification, measurements were focused on the different time points around 48 h which was when the leaf Ni contents exceeded those of the roots (i.e. 12, 24, 48, 72 h, and 1 week). In the roots, NA content was below the detection level in control plants. Upon Ni exposure, its amount increased regularly during the time-course of the experiment, reaching 15 nmol g–1 fresh weight (FW) after 1 week (Fig. 3C), in a similar way to what was already observed in the Ni dose–response experiment presented in Fig. 2C. In the leaves, in contrast, a transient decrease of the NA content was observed for the 24, 48, and 72 h time points, which reached its initial level after 1 week of treatment (Fig. 3C).
Time course of Ni abundance and identification of a stable NA–Ni complex in the xylem sap of Thlaspi caerulescens after nickel treatment
The results presented above strongly suggested a mobilization of the pool of NA from the leaves to supply the roots in order to cope with the Ni treatment. Furthermore, this leaf decrease coincided both with the increase of the amount of NA in the roots and with the transition of the plant Ni root-to-shoot ratio >1 (Fig. 3A), suggesting that NA could be involved in the translocation of Ni from roots to shoots. Therefore, this prompted determination of the Ni content in the xylem sap of plants subjected to the same Ni treatment. This concentration increased rapidly after 24 h of Ni treatment to reach a maximum of 400 µM, and then remained at the same level during the rest of the treatment (Fig. 4).
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The results strongly suggested a Ni-induced circulation of NA from the leaves to the roots and back to the leaves through the xylem sap. To determine if NA and Ni were associated during their circulation in the xylem sap, a procedure was used that was previously developed to identify the Ni-containing molecules in roots and leaves of plants treated with 100 µM NiSO4 (Ouerdane et al., 2006). This original approach was based on successive SEC and HILIC, enabling purification of traces of Ni-containing species that were then further identified by ESI-Q-TOF-MS/MS. The xylem sap samples collected 24, 48, 72 h, and 1 week after exposure to 100 µM NiSO4 were analysed using this analytical device. In the first chromatography (SEC), the Ni-containing molecule(s) eluted in a major and a minor peak with a retention time of 17 min and 15 min, respectively (Fig. 5A). In order to resolve the major Ni-containing fraction further, a second chromatography was performed, based on hydrophilic interactions (HILIC). During this chromatography, the major peak was also resolved in a single peak (data not shown). The fractions corresponding to this peak were collected off-line and analysed by ESI-Q-TOF-MS/MS to identify the organic ligand(s) involved in the chelation of Ni in the xylem sap. Observation of the mass spectra presented in Fig. 5B indicated a single Ni complex on the 200–450 m/z scale. It was identified as an Ni–NA complex by the characteristic isotopic pattern corresponding to the 58Ni (68%) and 60Ni (26%) isotopes with a
m of 1.9954 Da (Fig. 5B, insert). It was checked that below these masses no Ni pattern was observed and that above these masses peak intensities were too low (<1% of m/z 360). For m/z 304 and m/z 355–370, no ions corresponding to free NA, NA–Mn, NA–Cu, and NA–Zn were observed. The only heavy metal that can be observed by SEC-ICP-MS coupling in the same migration time as Ni–NA and that can enter into competition with Ni is Cu. This suspected Cu–NA complex corresponded only to 1–2% of the Ni–NA complex concentration and could not be observed by ESI-MS (data not shown). For all these reasons, NA appeared to be almost completely bound to Ni (>95%).
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From the SEC (Fig. 5A), it was also possible to estimate the amount of Ni–NA complex in the different xylem samples. Indeed, the first-dimension purification (SE-HPLC-ICP-MS) gave the amount of Ni found in a bound form, whereas the second dimension (HILIC-HPLC-ESI-MS/MS) identified NA as the unique chelator of Ni found in these peaks. As it is also known that the Ni–NA complex exists as one molecule of NA per molecule of Ni (Vacchina et al., 2003), it is therefore possible to estimate the amount of NA present in each Ni–NA-containing peak as equal to the amount of Ni. The results are presented relative to the total Ni content of the xylem sap (Fig. 6). At 24 h after the Ni treatment, the Ni–NA complex in the xylem represented 23% of the total Ni. Then the amount of the Ni–NA complex decreased slowly to reach a steady-state level of 8.5% after 1 week of exposure to Ni (Fig. 6).
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These results, together with the observation of an Ni-induced NA accumulation in the roots of T. caerulescens, a phenomenon absent in T. arvense (Fig. 2C), strongly suggested that NA might be involved in Ni translocation from the roots to the leaves in the metal hyperaccumulator T. caerulescens.
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NA is known to bind several metal ions in vitro (Stephan et al., 1996) and it has been previously shown that it also binds Ni in yeast cells expressing TcNAS (Vacchina et al., 2003). Furthermore, A. thaliana lines overexpressing TcNAS cDNA produce a large amount of NA, correlated to a better resistance to the toxicity of this metal (Pianelli et al., 2005). In the present study, the role of NA in T. caerulescens was analysed in response to Ni treatment. The genome of T. caerulescens appears to contain a single NAS gene (Fig. 1A). Although one copy is less than the four genes present in the A. thaliana genome (Suzuki et al., 1999), this situation is not unique since tomato also contains a single NAS gene whose mutation causes the classical Fe deficiency phenotype chloronerva (Ling et al., 1999). However, in contrast to a broad expression pattern of the NAS genes in various A. thaliana tissues (Suzuki et al., 1999), the TcNAS gene expression appeared restricted to the aerial part of T. caerulescens. This restricted expression is constitutive under the conditions tested, and is unaffected in response to Ni treatment (Figs 2B, 3B). Consistent with these expression data, the corresponding enzyme activity was also shown to be restricted to the leaves and independent of the Ni treatment. An amount of NA below the detection level in the roots (Figs 2C, 3C) does not appear to have deleterious effects. Indeed, the tomato chloronerva mutant depleted of NA is not affected in its root development (Pich and Scholz, 1993; Pich et al., 1994).
Upon Ni exposure, T. caerulescens displays an original behaviour. Although the TcNAS gene expression and the corresponding enzyme activity remain unchanged in response to Ni treatment, NA accumulates in the roots in a Ni dose-dependent manner (Fig. 2C). From a dynamic point of view, it is remarkable to observe that the increase of NA in the root in response to Ni as a function of time coincides with a transient decrease of NA content in leaves (Fig. 3C). For the 1 week time point, however, a 2-fold increase in the total amount of NA in the plants was observed, when comparing them with control plants. Several hypotheses can be proposed to explain this increase in the amount of NA at the whole plant level. Although the accumulation of the TcNAS mRNA was not significantly modulated in the leaves (Fig. 3B), an increase in mRNA stability could not be excluded. It is also possible that a slight increase in the NAS activity as observed in the leaves (Fig. 2D) is sufficient during a long period of Ni exposure to account for this NA increase. Another possibility concerns the stability of NA in the plant, which has not been documented thus far. Indeed, the half-life of NA could be modified, in this case increased, in the plant after Ni exposure. This stabilization of NA could also be responsible for the higher accumulation after 1 week. Nevertheless, these results suggest a mobilization of the foliar NA pool to furnish the roots upon Ni treatment, probably through the phloem sap. Such a phloem circulation of NA is well documented (Stephan and Scholz, 1993; Stephan et al., 1994; Schmidke and Stephan, 1995), but by the use of more suitable material, i.e. castor bean. In the case of T. caerulescens, the use of exogenous radiolabelled NA (not currently available) could help to visualize the circulation of this metabolite within the plant. Nevertheless, NA circulation through the phloem has already been proposed for normal mineral nutrition of plants (Stephan and Scholz, 1993; Stephan et al., 1994) as well as for the ‘normalization’ of the chloronerva phenotype by foliar application of NA (Stephan and Grün, 1985; Pich and Scholz, 1996).
Based on Ni measurement, the estimated Ni–NA concentration in the xylem sap of plants exposed to Ni suggests an increase in the amount of the complex in this sap (Fig. 6). This increased proportion of Ni bound to NA takes place during the first 24 h of Ni exposure and precedes the transition of the shoot-to-root Ni ratio to >1, which happens after 3 d (Fig. 3A). These results strongly suggest a direct link between the two phenomena. Another amino acid, histidine, has also been implicated in the response to Ni treatment in other plant species, Thlaspi goesingense, Alyssum lesbiacum, or Brassica juncea (Krämer et al., 1996; Kerkeb and Krämer, 2003). A correlation between the increase of Ni and of histidine in the xylem sap was shown in response to increasing concentrations of Ni, although the chelation of Ni by histidine has never been demonstrated directly and this complex was never detected in T. caerulescens.
The present results have clearly identified NA as a chelator of Ni during its translocation from the root to the shoot through the xylem sap of T. caerulescens. The relative proportions of Ni–NA complex measured in the xylem sap indicate an excess of Ni compared with NA (Fig. 6). It could not be excluded that other Ni chelates could exist, but that were too weak to be visualized under the present conditions. However, using the same analytical procedure, citrate, malate, and histidine (in addition to NA) were already identified as Ni chelators in T. caerulescens root and leaf tissues (Ouerdane et al., 2006) indicating that if they were involved in Ni chelation in the xylem sap, they would have been detected. The difference in xylem concentrations between Ni and NA (only between 8.5% and 23% of Ni bound to NA) could have two explanations. First, NA would be just involved in the chelation of Ni, representing no more than 10% of the Ni circulating in the xylem. Given the characteristics of the xylem, Ni could possibly circulate as a free hydrated ion. In that case, one could hypothesize another role for NA in the loading and/or the unloading of the xylem. Accordingly, there would be no need to reach equimolar concentrations of NA and Ni in the xylem sap. Such a dynamic system of circulation of NA would require the association and dissociation of Ni–NA complexes. Indeed, concerning Fe, it has been hypothesized that NA could play the role of a shuttle by chelating Fe2+ from ITP-bound Fe3+ during loading and unloading of the phloem sap (Krüger et al., 2002; Curie and Briat, 2003).
The use of naturally metal-tolerant plant species has enabled the identification of an original response to Ni in T. caerulescens. In the roots, a concomitant accumulation of both Ni and NA is observed upon exposure to different concentrations of Ni, although no TcNAS gene expression and no TcNAS enzyme activity occur in this organ. The formation of Ni–NA complexes in vivo, in the T. caerulescens xylem sap, was demonstrated by the use of hyphenated techniques (HPLC-ICP-MS and HPLC-ESI-MS). Such a mechanism is absent in the metal-sensitive species T. arvense where there is neither NA accumulation in the roots, nor Ni translocation to the leaves upon Ni exposure. This results in a massive and potentially toxic Ni accumulation in the roots of this plant. The transport of NA and Ni–NA complexes within the plant needs crossing of membranes through specific transport systems. YS1-like proteins (YSLs), proposed to be potential metal–NA transporters in A. thaliana (Curie et al., 2001), could be such candidate transporters, and are therefore important targets to characterize. For that reason, three YSL genes have been cloned from T. caerulescens, homologous to AtYSL3, 5, and 7, and it was shown that at least TcYSL3 is able to transport Ni–NA and Fe–NA complexes (Gendre et al., 2006). This work provides, at the molecular level, new insights in metal chelation and translocation mechanisms, and original targets for biotechnological manipulation of metal tolerance and accumulation in plants.
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Acknowledgements |
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We thank Mr Guy Delmot for providing the seeds of Thlaspi caerulescens, and Gabriel Krouk for sharing the 25S probe. The work of DG was supported by a thesis fellowship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie. This work was supported by Centre National de la Recherche Scientifique (CNRS), by Institut National de la Recherche Agronomique (INRA), and by the Toxicologie Nucléaire Programme (ToxNucE) of the Réseau Inter-Organismes (RIO). UMR Biochimie et Physiologie Moleculaire des Plantes is LRC CEA number 20V.
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Abbreviations |
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ESI-MS, electrospray ionization-mass spectrometry; HILIC, hydrophilic interaction liquid chromatography; HPLC, high-pressure liquid chromatography; ICP-MS, induced coupled plasma-mass spectrometry; ITP, iron transport protein; NA, nicotianamine; NAS, nicotianamine synthase; SEC, size exclusion chromatography; SE-HPLC, size exclusion-HPLC; TOF, time-of-flight; YS-1, yellow stripe 1; YSL, YS-1 like.
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