JXB Advance Access published online on November 25, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern286
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RESEARCH PAPER |
Nicotianamine and histidine/proline are, respectively, the most important copper chelators in xylem sap of Brassica carinata under conditions of copper deficiency and excess
Dipartimento di Chimica e Biotecnologie Agrarie, Università di Pisa, via del Borghetto 80, 56124 Pisa, Italy
* To whom correspondence should be addressed: E-mail: irtellibarbara{at}yahoo.it
Received 30 September 2008; Revised 16 October 2008 Accepted 20 October 2008
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Abstract |
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The effect of two different copper conditions (deficiency and excess) on the amino acid composition in B. carinata xylem sap was analysed. When the Cu in the nutrient solution was increased from 0.12 to 2.5 or 5 µM, the concentrations of histidine, threonine, glutamine, proline, methionine, and glycine were much increased in the xylem sap. When Cu was made deficient in the nutrient solution by decreasing its concentration from 0.12 µM to 0 µM, nicotianamine, glutamine, and threonine were significantly increased in the xylem sap. Aqueous solutions containing different Cu–amino acid complexes (simulated saps) responded in a specific way to the changes in pH, providing a signature that was used to evaluate, by comparison with the real xylem sap, the importance of each amino acid in the xylem transport of Cu. For a single amino acid, the free solution Cu2+ concentration versus pH titration curves for histidine and proline were the most similar to that for xylem under Cu excess. Under Cu deficiency, this Cu concentration versus pH titration curve appeared to be very similar to that for nicotianamine. It is concluded that increased Cu concentrations induced the selective synthesis of certain amino acids in the sap, of which histidine and proline are the most important. Under Cu deficiency, the concentration of nicotianamine was induced the most. The fact that nicotianamine is induced under Cu starvation and not under Cu excess, is in contrast to similar studies indicating species-specific reactions. However, the induction of nicotianamine under Cu starvation is in line with recent molecular data of the role of nicotianamine in intracellular Cu delivery.
Key words: Brassica carinata, copper, histidine, nicotianamine, proline, xylem sap
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Introduction |
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The average copper concentration in the Earth's crust is about 70 mg kg–1 soil, but in the vicinity of anthropogenic activities copper concentrations can increase dramatically (Fregoni and Corallo, 2001; Pacyna, 2002).
Although copper is an essential element for plants, animals, and human beings it can be toxic above a certain concentration. Copper's primary action in plants takes place on the root plasma membranes (Quartacci et al., 2001), but when present at toxic concentrations it may interfere with numerous physiological processes in leaves through inhibitory effects on photosynthetic electron transport and the degradation of the chloroplast inner structure and pigment content (Ciscato et al., 1997; Horváth et al., 1998; Navari-Izzo et al., 1998, 1999; Quartacci et al., 2000).
A thorough understanding of Cu uptake and translocation via the xylem fluid to plant shoots will assist in solving the problems linked to Cu toxicity in the upper plant parts. Metals like copper are highly reactive in biological systems, so long-distance movement probably involves the chelating of metals in order to be non-reactive. This hypothesis has been confirmed by several studies. Cu was present in the xylem sap of four dicotyledonous plants in negatively charged complexes possibly involving amino acids (Tiffin, 1972). Moreover, more than 99% of total Cu was in a complexed form in sunflower xylem sap (Graham, 1979) and similar results were found for chicory and tomato xylem saps in a recent study by Liao et al. (2000a).
Although other organic compounds, such as citrate, can ligate copper in xylem sap (Mullins et al., 1986), almost all investigations have been focused exclusively on amino acids. Amino acids are present in relatively high concentrations in xylem sap normally higher than the total Cu concentration, and can form very stable complexes with Cu (May et al., 1977). White et al. (1981a, b) suggested that Cu was bound to several amino acids, mostly asparagine (Asn) and histidine (His) in soybean exudates and His, glutamine (Gln), and Asn in tomato exudates. Asn and His were also predicted to be the major ligands for Cu in soybean and tomato xylem saps by Mullins et al. (1986). Luckily, due to the improved technologies and the implementation of molecular biological methods, recent in vivo studies have made it possible to identify the most important long-distance Cu-chelating compounds. They appeared to be all amino acids, confirming the earlier results of the simulation models (Herbik et al., 1996; Pich and Scholz, 1996; Liao et al., 2000b; Takahashi et al., 2003; Kim et al., 2005).
Among all the compounds investigated, the non-proteinogenic amino acid nicotianamine (NA) seems to play a key role in copper complexation in plants xylem sap (Herbik et al., 1996; Pich and Scholz, 1996; Liao et al., 2000b; Takahashi et al., 2003; Kim et al., 2005). Much of our understanding of the physiological functions of NA comes from the complex phenotype of a tomato mutant called chloronerva. The chloronerva mutation (chl) disrupts the function of the single gene encoding NA synthase (Herbik et al., 1996). Homozygous chl plants contained excess copper in roots, but failed to transport normal amounts of copper into mature leaves. Furthermore, xylem exudates from mutant plants showed unusually low levels of copper (Herbik et al., 1996; Pich and Scholz, 1996). These results were confirmed by Liao et al. (2000b) who reported that NA is likely to be the most important Cu ligand in tomato and chicory xylem exudates. However, the xylem transport of Cu in tomato and chicory was efficient even in the absence of NA, provided that His was present (Liao et al., 2000b). Notably, as far as the stability constant is concerned, His (logKst=17.5) could compete with NA (logKst=18.6) as a ligand for Cu.
The objective of the present study was to investigate the effect of two different copper conditions (Cu excess and Cu deficiency) on the amino acid composition in B. carinata xylem sap. The importance of each amino acid in the xylem transport of Cu was therefore evaluated using the sensitivity to the solution pH of the Cu-complexes containing carboxylic functional groups (Liao et al., 2000b). In fact, aqueous solutions containing different Cu–amino acids complexes (simulated saps) responded to changes in the solution pH in a specific way, providing a signature that was used to evaluate, by comparison with the real xylem sap, the participation of each amino acid in the xylem transport of Cu.
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Chemicals
Water was obtained from a Milli-Q purification system and had a resistance greater than 18.2 M
cm–1. Copper sulphate, all amino acids used as standards, all buffers, and reagents for cytosolic contamination analyses were obtained from Sigma-Aldrich Co. (St Luis, MO, USA). (–)-Nicotianamine was obtained from T Hasegawa Co., Ltd (Kawasaki-shi, Japan). All chemicals used were analytical grade reagents.
Plant culture
Seeds of Brassica carinata cv. 079444 were surface-sterilized for 10 min with diluted NaClO (about 2% active chlorine). After germination in the dark (3 d), seedlings were transferred in plastic pots (10 plants per pot) containing 4.0 l of nutrient solution (0.25 mM NH4H2PO4, 1 mM Ca(NO3)2, 0.5 mM MgSO4, 1.5 mM KNO3, 11.5 µM H3BO3, 5 µM Fe2(C4H4O6)3, 3.5 µM MnCl2, 0.03 µM MoO3, 0.3 µM ZnSO4, and 0.12 µM CuSO4, pH 5.5). For nutrient solution preparation, the precaution was taken to avoid the use of salts containing Cu as a contaminant. Plants were grown for 3 weeks in a controlled environment growth cabinet (day/night period 16/8 h, day/night temperature 22/20 °C, relative humidity 75%, and light intensity 400 µmol m–2 s–1 photon flux density). The nutrient solution was changed three times every week and aerated continuously. Three weeks after germination, plants were removed from the nutrient solution and transferred to the treatment solutions. The experimental set-up was arranged in a completely randomized block design with three replicate blocks per copper concentration. Three independent experiments were performed. Plants were further grown for 3 d in a modified nutrient solution containing 0, 2.5, and 5 µM Cu supplied as CuSO4. The normal nutrient solution (0.12 µM CuSO4) was the control. The pH of the culture solutions was adjusted to 5.5 with KOH. The pH and copper concentration of the culture solutions were monitored daily and the solutions were renewed every day.
Xylem sap collection
At harvest, B. carinata stems were cut using a stainless-steel razor blade at about 2 cm above the media surface perpendicular to the stem axis. To avoid contamination of the xylem exudates with the cell sap, the first drop of exudate was discarded. The plants remained in the solutions. The decapitated stumps were wiped gently and fitted with Tygon tubing. Since the composition of the xylem sap is known to change with time (Hocking et al., 1978), the stems were always cut between 17.00 h and 17.30 h. The xylem sap was collected during the night in the dark at 20–25 °C. The saps were filtered (0.45 µm) and frozen at –80 °C until needed. Each collection unit consisted of one plant.
Xylem sap analysis
The xylem pH was measured directly with a Metrohm 654 pH-meter (Metrohm LTD, Herisau, SW). The total Cu concentration in the xylem sap was measured by direct aspiration of the xylem sap using an atomic absorption spectrophotometer (Model 373, Perkin-Elmer, Thornill, ON, Canada). Free Cu2+ concentrations were measured with a cupric electrode (Orion 9629BN IonplusTM Cupric Electrode; Thermo Orion, Beverly, MA, USA) which only responds to Cu2+ ions in solution.
Contamination of the xylem saps by cytosolic enzymes was assessed in each sample as in Lòpez-Millàn et al. (2000). Cytosolic malate dehydrogenase (c-mdh; EC 5.3.1.9 [EC] ) and cytosolic hexose phosphate isomerase (c-hpi; EC 1.1.1.37 [EC] ) were used as cytosolic contamination markers for the xylem sap. The activity of these two markers in the xylem sap was checked against the corresponding activities in plant tissue homogenates. Plant tissues were homogenized with 2 ml of a buffer (pH 8) containing 100 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid], 30 mM sorbitol, 2 mM dithiothreitol, 1 mM CaCl2, 1% (w/v) bovine serum albumin, and 1% polyvinylpyrrolidone. The supernatant was collected after 10 min centrifugation at 10 000 g and analysed immediately. The activity of c-mdh was determined using oxalacetate as substrate and measuring the decrease in A340 due to the enzymatic oxidation of NADH. The final reaction mixture (pH 9.5) was 46.5 mM TRIS [tris(hydroxymethyl)-aminomethane], 0.1 mM NADH and 0.4 mM oxalacetate. The activity of c-hpi was determined using Fru-6-P as the substrate, which is converted by c-hpi into Glu-6-P. This is then oxidized by exogenous glucose-6-phosphate dehydrogenase and the simultaneous reduction of NADP+ was measured from the increase in A340. The final reaction mixture (pH 8) was 50 mM TRIS, 5 mM MgCl2, 1 mM NaCl, 0.4 mM NADP+, 0.46 U ml–1 glucose-6-phosphate dehydrogenase, and 1.4 mM Fru-6-P.
Amino acid detection in B. carinata xylem saps
Major amino acids in xylem exudates of B. carinata were separated by reversed phase high performance liquid chromatography (RP-HPLC) and quantified by UV detection. Before separation, amino acids were converted into their phenylthiocarbamyl (PTC) derivatives (Gonzàlez-Castro et al., 1997). 0.5 ml of standard solutions or B. carinata xylem saps were transferred to a flask and vacuum-dried at 40 °C. 0.5 ml of methanol/water/triethylamine (2:2:1 by vol.) was added to the residue and then vacuum removed at 40 °C. Next, 0.5 ml of the derivatizing reagent methanol/water/triethylamine/phenylisothiocyanate (7:1:1:1 by vol.) was added, the flask was shaken at room temperature for 20 min. Finally, the solvents were removed under a nitrogen stream and the flask was sealed and stored at 4 °C. Pending analysis, the residue was resuspended in 1 ml of eluent A. Amino acids were separated on a C18 Waters Spherisorb ODS2 column (4.6x250 mm; 5 µm; spherical) and detected at 254 nm wavelength by Waters UV 2487. The column temperature was controlled at 40±0.5 °C using a Recipe HPLC Thermostat HT 300. The sample injection volume was 20 µl. Eluent A was an aqueous buffer prepared by adding 0.5 ml l–1 of triethylamine to 0.14 M ammonium acetate and titrating it to pH 6.2 with glacial acetic acid. Acetonitrile/water/isopropanol 60:38:2 (by vol.) was used as eluent B. The gradient program employed for the separation of PTC-amino acids is adequately described by Gonzàlez-Castro et al. (1997). The separation between threonine (Thr) and 
-aminobutyric acid (GABA) was obtained by running a separate chromatogram in which the column temperature was set at 25 °C.
The retention times of amino acids were checked using authentic standards and their quantification was obtained from the relationship peak area versus concentration of standards. Chromatogram analysis was performed by Millennium 32 (Waters).
Free Cu2+ concentration versus pH titration
The stability of Cu-complexes containing carboxylic acid functional groups is normally sensitive to solution pH. The proportion of free Cu2+ ions varies with solution pH providing a ‘signature’ for the Cu-organic complex system (Liao et al., 2000b). A cupric electrode was used to measure the free Cu2+ ion concentration in amino acid solutions, plant xylem saps, and simulated saps at varying solution pH values. Xylem sap (5 ml) collected from B. carinata treated with 5 µM CuSO4 was transferred into a 10 ml vial and its pH was set at 6.5. The pH of the solution was then decreased stepwise from 6.5 to 3 while the solution free Cu2+ concentrations and pH were monitored. Solution of 10 mM KOH and HNO3 were used to adjust the pH. Since the total volume of these adjustments represented less than 0.4% of the total solution volume, no corrections for dilution were made.
Thereafter, in order to establish the importance of each amino acid detected in the B. carinata xylem in Cu transport, simulated xylem saps were prepared and titrated as described above. The simulated saps were prepared by adding to deionized water a single amino acid or a combination of amino acids and copper to the concentration of real xylem sap from plants grown in 5 µM Cu2+ (i.e. 0.94 µmol l–1 xylem).
The xylem sap of plants treated with 0 µM CuSO4 had a xylem copper concentration that was too low to be detected by the cupric electrode (0.16 µmol l–1 total xylem Cu concentration). So, an exogenous amount of copper was added to the xylem sap to make the concentration 0.94 µmol l–1 xylem. The titration of the modified xylem sap was conducted as previously described. Also in this case, simulated xylem sap containing single amino acid were prepared and titrated.
Statistical analysis
Results are the means ±SD of three replicates of three independent experiments. Since no significant differences were detected among the three experiments, the number of replicates (n) was considered to be 9. Significance was calculated using Tukey Test (P 
0.01).
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Results |
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Xylem sap analysis
The xylem pH (5.8±0.6) of Brassica carinata was independent of the amount of copper present in the culture solutions.
The cytosolic marker enzymes (malate dehydrogenase, c-mdh, EC 5.3.1.9
[EC]
; hexose phosphate isomerase, c-hpi, EC 1.1.1.37
[EC]
) were always 1% of those found in total plant tissue homogenates indicating that no appreciable cytosolic contamination was present in B. carinata xylem sap.
Determination of amino acids in B. carinata xylem sap
Xylem saps of B. carinata plants grown in nutrient solution (0.12 µM CuSO4; control) or incubated for 3 d in treatment solutions (0, 2.5 or 5 µM CuSO4) were collected. In the xylem sap of B. carinata 21 amino acids were found (Table 1). Under control conditions, nicotianamine (NA) was the major amino acid present in xylem sap (64.62 µmol l–1 xylem) followed by lysine (Lys) (51.71 µmol l–1 xylem), alanine (Ala) (43.66 µmol l–1 xylem), leucine (Leu) (22.18 µmol l–1 xylem), histidine (His) (18.63 µmol l–1 xylem), and valina (Val) (16.55 µmol l–1 xylem) (Table 1).
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Compared with the control (CuSO4 0.12 µM; Table 1), the amino acid concentrations in the xylem sap showed some significant changes if B. carinata plants were exposed to 2.5 and 5 µM CuSO4 (Table 1). The amino acids which showed the greatest relative increases in concentration when B. carinata was exposed for 3 d to 2.5 µM and 5 µM CuSO4 were: histidine (His) (+452% and +641%, respectively), threonine (Thr) (+292% and +398%, respectively), glutamine (Gln) (+118% and +250%, respectively), proline (Pro) (+116% and +135%, respectively), methionine (Met) (+82% and +108%, respectively), and glycine (Gly) (+82% and +89%, respectively) (Table 1).
Compared with the control (CuSO4 0.12 µM, Table 1), the xylem amino acid concentrations showed some significant changes when B. carinata plants were grown for 3 d under copper deficiency (CuSO4 0 µM, Table 1). The amino acids that showed significant relative increases in concentration when B. carinata was grown under copper deficiency were: methionine (Met) (+390%), nicotianamine (NA) (+320%), glutamine (Gln) (+318%), and threonine (Thr) (+302%) (Table 1).
Total Cu in xylem sap
Total Cu concentration in B. carinata xylem sap grown under control condition (0.12 µM CuSO4) was 0.32 µmol l–1 xylem. When the Cu concentration in the nutrient solution was increased from 0.12 µM to 2.5 µM and 5 µM, the concentration of Cu was much increased (0.63 µmol l–1 xylem and 0.94 µmol l–1 xylem, respectively). When Cu was made deficient in the nutrient solution (0 µM CuSO4), the concentration of xylem Cu decreased to 0.16 µmol l–1 xylem.
Free solution Cu2+ concentration versus pH titration curves for Brassica xylem sap and simulated sap in the case of Cu excess
Increased Cu concentration in the nutrient solution induced the selective accumulation of certain amino acids in the xylem sap. The plant response, in terms of amino acid accumulation, was directly correlated with the copper concentration. In fact, the 2.5 µM and 5 µM Cu treatments induced the accumulation of the same amino acids although to different extents. For this reason, the free Cu2+ concentration versus pH titration signatures were conducted only for plants treated with 5 µM CuSO4 (Fig. 1).
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The free Cu2+ concentrations versus pH titration curves of B. carinata xylem sap and the key amino acids detected in the xylem sap are presented in Fig. 1. For a single amino acid, the pattern of the titration curve of 139.99 µmol l–1 histidine (His) (Fig. 1A) with 0.94 µmol l–1 Cu was the most similar, closely followed by that of 6.34 µmol l–1 proline (Pro) (Fig. 1C), to the curve of B. carinata xylem.
In order to investigate the relative importance of histidine and proline on Cu binding in the presence of other amino acids, titration experiments were conducted on the following solutions: B. carinata xylem sap (Fig. 1); simulated xylem sap (His+Thr+Gln+Gly+Pro+Met) (Fig. 1D); simulated xylem sap without histidine (Thr+Gln+Gly+Pro+Met) (Fig. 1D); simulated xylem sap without proline (His+Thr+Gln+Gly+Met) (Fig. 1D). The pattern of Cu2+ release from Cu-complexes of the simulated xylem sap without histidine (Fig. 1B) was the most different from the pattern of B. carinata xylem sap, indicating that histidine is probably the most important copper chelator in the xylem sap of B. carinata grown for 3 d in excess copper.
In the xylem sap of B. carinata treated with copper excess (5 µM CuSO4), 139.99 µmol l–1 histidine (His), 21.74 µmol l–1 threonine (Thr), 7.39 µmol l–1 glutamine (Gln), 22.91 µmol l–1 glycine (Gly), 6.34 µmol l–1 proline (Pro), and 12.53 µmol l–1 methionine (Met) could account, at pH 5.8±0.6 (B. carinata xylem sap pH), for 67.4, 41.6, 45.3, 52.1, 59.5, and 32.7% complexation of 0.94 µmol l–1 Cu, respectively (Fig. 1A, B, C; Table 2). A combination of six key amino acids (His+Thr+Gln+Gly+Pro+Met) could account for 69.9% complexation of 0.94 µmol l–1 Cu, which is very similar to the measured level complexation in B. carinata xylem sap (72.7%) (Fig. 1D; Table 2). Simulated xylem sap without histidine (Thr+Gln+Gly+Pro+Met) and simulated xylem sap without proline (His+Thr+Gln+Gly+Met) could account for 59.2% and 68.4% complexation of 0.94 µmol l–1 Cu, respectively (Fig. 1D; Table 2).
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Effect of excess of external Cu on the concentration of histidine and proline in xylem sap
The histidine content increased with increasing external Cu concentration. Regression analyses showed that the concentration of histidine in B. carinata xylem saps was linearly related with the external copper concentration (y=7.07x where y=[His]xyl and x [Cu]ext; R2=0.88) and xylem Cu concentration (y=463x–5.1 where y=[His]xyl and x=[Cu]xyl; R2=0.95). On the other hand, proline only showed a significant increase with the 2.5 µM CuSO4 treatment since no significant differences were detected between the contents of proline at the 2.5 µM and 5 µM CuSO4 treatments (Table 1).
Free solution Cu2+ concentration versus pH titration curves for Brassica xylem sap and simulated sap in the case of Cu deficiency
Copper deficiency induced the selective accumulation of methionine, nicotianamine, glutamine, and threonine in the xylem sap.
The free Cu2+ concentrations versus pH titration curves of B. carinata xylem sap and key amino acids detected in xylem sap are presented in Fig. 2. When singly applied, glutamine (Gln), methionine (Met), and threonine (Thr) showed titration patterns very different from that of the xylem fluid of B. carinata (Fig. 2A, C, D). Conversely, the 269.99 µmol l–1 nicotianamine (NA) pattern appeared to be very similar to that of B. carinata xylem sap (Fig. 2B).
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Discussion |
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Numerous studies have been conducted to identify the most important chelating agent of copper in the xylem sap. Simulation models (White et al., 1981a, b; Mullins et al., 1986) and, more recently, in vivo studies (Herbik et al., 1996; Pich and Scholz, 1996; Liao et al., 2000b; Takahashi et al., 2003; Kim et al., 2005) irrefutably show that the most important long-distance Cu-chelating compounds are amino acids.
Because this is the first study conducted on B. carinata, the amino acids concentrations (Table 1) were compared with those found previously for other plant species and it appeared that the concentrations were of the same order of magnitude or one order less (White et al., 1981b, Senden et al., 1992; Krjiger et al., 1999; Liao et al., 2000b). One might, therefore, conclude that the xylem amino acid content of plants grown in optimal conditions does not vary significantly. Conversely, the amino acid composition seems to be species-dependent, although, in most cases, histidine and nicotianamine (if analysed) are two of the most common amino acids in xylem sap. In the present experiments, nicotianamine was the major amino acid present in B. carinata xylem sap followed by lysine, alanine, leucine, histidine, and valine (Table 1). Nicotianamine, a phytosiderophore, was recently detected in tomato and chicory xylem saps (Liao et al., 2000b) and was also found in earlier studies (Noma et al., 1971; Noma and Noguchi, 1976; Budê
ínsk
et al., 1980; Herbik et al., 1996; Pich and Scholz, 1996) confirming the widespread occurrence of this amino acid in higher plants.
When exposed to high Cu concentrations in the nutrient solution, the relative increases in the concentration of certain amino acids are higher than others. Notably, histidine, which can form complexes with Cu with high association constant (logKst=17.5), was the amino acid with the greatest relative increase (Table 1) followed by others with lower stability constants (May et al., 1977). Surprisingly, an almost constant content of nicotianamine, the amino acid with the highest stability constant (logKst=18.6) and the highest absolute concentration in Brassica xylem sap, was detected during both copper excess treatments (Table 1). This is in contrast with Liao et al. (2000b) who found that both nicotianamine and histidine were the amino acids with the greatest increments when tomato and chicory plants were exposed to high copper concentrations. Although it is not possible to ascribe these differences to a particular factor, it is reasonable to consider these discrepancies constitutive at species level rather than imposed by different treatment levels or general growth conditions. In fact, B. carinata seems to respond to high copper concentrations inducing the synthesis of species-specific amino acids as shown by preliminary experiments in which plants are treated with copper concentrations much higher than those used in the present work (data not shown).
Under copper deficiency conditions, methionine, nicotianamine, glutamine, and threonine were detected as the amino acids with the highest relative increases (Table 1). Since the production of free amino acids in the xylem of plants as a response to copper starvation has not previously been reported, it is not possible to confirm the data obtained by comparing them with others. It seems that the response to copper stress is not only species-specific but also stress-specific since the accumulation of different amino acids in the xylem sap occurred in the same plant species, B. carinata, exposed to different stress conditions.
The pH-sensitive Cu-binding signature curves of B. carinata xylem sap and simulated saps containing single amino acids (Fig. 1) showed that histidine and proline are likely to be the most important copper ligands in xylem exudates of B. carinata treated with excess copper. The titration of simulated saps containing combinations of amino acids (Fig. 1), confirmed the role of histidine as the major copper chelator, although it was not the only one.
In the absence of histidine, proline would play a very important role in Cu binding (Fig. 1; Table 2). The role of threonine (21.74 µmol l–1), glutamine (7.39 µmol l–1), glycine (22.91 µmol l–1), and methionine (12.53 µmol l–1) were negligible in the presence of 139.99 µmol l–1 histidine and 6.34 µmol l–1 proline (Fig. 1; Table 2). Although these results are in contrast to those of White et al. (1981a) who indicated that Cu was primarily bound to asparagine, glutamine, and histidine, they are partially confirmed by the use of theoretical speciation models (Visual MINTEQ ver 2.53; Gustafsson, 2006). Based on the stability constant of the Cu-(glycine)x complexes (where x=1 or 2) and the concentration of copper and glycine found in the xylem sap of B. carinata treated with 5 µM CuSO4, theoretically it was possible to calculate the amount of free copper (52.6%) at pH 5.8 (xylem sap pH). This result is in agreement with what was found experimentally in the free Cu versus pH titration curve of glycine (47.9%) (Table 2; Fig. 1). In addition, the presence of an about 30% of free copper in B. carinata xylem sap (Table 2) is reasonable at pH 5.8 since the amino acids are, at that pH, partially protonated and therefore not available for complexation. This explains the discrepancies between the present study and previous work that found more than 90% of copper in a complexed form (Graham, 1979; Liao et al., 2000a).
Cations, especially the heavy metals Co, Ni, and Zn, may affect Cu speciation because of a competition of those metals with copper for the same ligands. However, Co and Ni were not detectable in B. carinata xylem saps and the Zn concentrations in the xylem saps of Brassica plants receiving normal Zn supply were very low (data not shown). Such low concentrations do not affect the Cu complexation by histidine and proline because of the much lower association constants with histidine and proline compared with Cu (May et al., 1977). Additional analysis of xylem sap from B. carinata incubated for 3 d in excess copper treatments (2.5 µM and 5 µM) showed consistent increases in histidine and proline as the copper concentration increased in solution. In particular, the histidine concentration increased in direct proportion to the external Cu concentration. However, the histidine and proline concentrations were always high enough to account for all the complexed Cu in xylem sap.
Although in cases of copper deficiency, the pH-sensitive Cu-binding signature curves were performed on xylem saps to which exogenous copper had been added (Fig. 2), it is not unreasonable to suggest that the same trends would exist in native xylem saps.
The pH sensitive Cu-binding signatures curves of B. carinata xylem sap and simulated saps containing single amino acids (Fig. 2) showed that nicotianamine is probably the most important copper ligand in xylem exudates of B. carinata under conditions of copper deficiency.
Nicotianamine had already been identified and quantified in plant xylem sap and suggested as a possible copper ligand (Herbik et al., 1996; Pich and Scholz, 1996; Liao et al., 2000b). Herbik et al. (1996) showed that the nicotianamine-deficient mutant chloronerva of tomato suffers from Cu deficiency in the shoots. These results were confirmed by Liao et al. (2000b). They reported that NA is likely to be the most important Cu ligand in tomato and chicory xylem exudates.
Another important enzyme in the regulation of the level of nicotianamine is the nicotianamine aminotransferase (NAAT) that catalyses the amino group transfer of NA in the biosynthetic pathway of phytosiderophores. The gene that encodes NAAT from barley was introduced into the non-graminaceous plant, tobacco. Transgenic tobacco plants (naat tobacco) showed a lower concentration of Cu, Mn, Fe, and Zn in both young leaves and flowers than the wild type. This decrease, attributable to the depletion of endogenous NA, is a further confirmation of the role of NA in metals transport (Takahashi et al., 2003).
The increase in nicotianamine detected in conditions of copper starvation but not in copper excess seems to indicate that this non-proteinogenic amino acid is not involved in the response to excess of copper but participates in Cu transport to the shoots in conditions of deficiency. This could be explained when the biosynthetic pattern of nicotianamine is taken into consideration. Nicotianamine is an intermediate in mugineic acid (MAs) biosynthesis formed via the nicotianamine synthase-catalysed trimerization of S-adenosyl-L-methionine. Mugineic acids are naturally secreted from graminaceous plants in iron starvation conditions in order to solubilize Fe in the soil. Unlike MAs, nicotianamine is not secreted from the roots but it is not unreasonable to think that its increase is involved in internal copper transport when the plant is in metal deficiency rather than in metal excess.
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The authors thank Professor Jos Verkleij (Vrije Universiteit, Amsterdam) for the useful suggestions and critical readings of the manuscript. Professor Marco Mazzoncini (University of Pisa) kindly provided seeds of B. carinata cv. 079444. This study was funded by the University of Pisa (Fondi di Ateneo, 2005) and by MIUR (Cofinanziamento, 2005).
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