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Plant cultivation and harvest
Seeds of T. arvense (collected in Nancy, France), A. murale (collected in Pojska, Albania) and N. caerulescens (collected in Puy de Wolf, France) were sown on agar and germinated in the dark at 25 °C for 5 days. Then 24 seedlings of each species were transferred to 5 L nutrient solutions in a growth chamber for pretreatment. The solution for T. arvense contained the following nutrients (in µM): 1000 Ca(NO3)2, 1000 KNO3, 500 MgSO4, 100 KH2PO4, 50 KCl, 10 H3BO3,1 MnCl2, 0.2 CuSO4, 0.2 Na2MoO4, 5 Fe(III)-EDTA, 2 NiSO4 and 2 ZnSO4. Two mM 2- morpholinoethanesulphonic acid (MES) was used to buffer the pH, which was adjusted to 5.8 by the addition of 1 M KOH. The nutrient solution used to cultivate A. murale and N. caerulescens was based on the previous one, with a lower Ca/Mg ratio, to mimic the soil conditions of serpentine areas where the plant seeds were co llected; the Ca and Mg concentrations were 500 and 1000 µM, respectively. The growth conditions were 22/18 °C day/night temperatures, 70% relative humidity, 16 h photoperiod and 150 µ mol s−1 m−2 light intensity.
After 14 days of pretreatment, the seedlings were transferred to 2 L containers and treated with low and high levels (2 and 50 µM) of Ni and Zn nutrient solutions. The treatments were (Ni/Zn sulfate in µM/µM): T.a. 2/2 (T. arvense), A.m. 50/2 (A. murale), A.m. 50/50 (A. murale), N.c. 2/2 (N. caerulescens), N.c. 50/2 (N. caerulescens) and N.c. 50/50 (N.
Isotope fractionation of Ni in hyperaccumulators
caerulescens). To avoid iron deficiency, 20 µM instead of 5 µM of Fe(III)-EDTA was used in 50/2 and 50/50 treatments. Each treatment replicated three times and each contained one plant. The solutions were renewed weekly during the first two weeks and then twice a week.
Plants were harvested after 12 d (for T. arvense) or 28 d (for A. murale and N. caerulescens) of treatment. Roots were soaked in 1 mM LaCl3 and 0.05 M CaCl2 solution for 15 min at 0°C to remove the Ni and Zn adsorbed on the root surface (Weiss et al. 2005). The plants were washed by ultrapure water (Millipore, 18.2 MΩ cm-1), then separated into root, stem and leaf (for T. arvense and A. murale), or root and shoot (for N. caerulescens, which was at the rosette stage), and later dried at 70 °C for 3 days. The dry samples were ground to fine powders (0.5 mm sieve) for analysis.
All the harvested plant samples (between 3.4 to 95.8 mg) were placed in Teflon beakers and digested by 5 ml of concentrated HNO3 on a hot plate. After digestion, the solutions were evaporated to dryness and the residues were dissolved by 1 mL of 0.1 M HNO3. The Ni and Zn concentrations were determined by ICP-MS (Perkin-Elmer ICP-MS SCIEX Elan 6000 or Thermo X7). To evaluate blank contribution, a procedural blank was introduced into each sample series. The average blank measured throughout the study was 35 ± 5 ng of Zn (n=3), which is negligible compared to the Zn contents in samples (7 – 230 μg). Ni in the blanks was under the determination limit (< 0.9 ng).
Ni and Zn purified fractions for isotope analyses were recovered simultaneously from the same aliquot of sample. The Zn purification method for the column chemistry was adapted from Cloquet et al. (2006), while Ni method from Quitte and Oberli (2006) and Gueguen et al.(2013). Initially, a sample which contained 2 μg Ni was equilibrated overnight with 2.3 μg of double-spike (mixture of equivalent amount of 61Ni and 62Ni, bought from Oak Ridge National Laboratory). After evaporation, the residue was dissolved in 1 ml of 6 M HCl. The sample was loaded onto 2 ml AG1-X8 (Bio-Rad) resin bed. At this step of the chemistry, Zn was fixed in the resin bed and could be eluted. Fe was removed using 10 ml of 0.5 M HCl, while Zn fraction was recovered by elution of 10 ml of 0.5 M HNO3. The latter purification step was repeated twice.The loading and rinsing solutions from the first column (15 ml of 6 M HCl) were collected and dried for the second step of purification for Ni. The dried residue was dissolved in 1 ml of 1 M HCl, mixed with 0.3 ml of 1 M ammonium citrate and the pH of the mixture was adjusted to 8 – 9 by adding NH4OH. Then, sample was loaded onto 0.5 ml resin bed of a Ni-specific resin containing dimethylglyoxime (DMG) as Ni-chelating agent. Beforehand, the resin was conditioned in ultrapure water and 2 ml of 0.2 M ammonium citrate (pH 8 – 9), which was used for matrix elution as well (4 ml). After rinsing, Ni was eluted by 4 ml of 3 M HNO3. The eluting solution was dried and 5 ml of concentrated HNO3 was added to break down DMG complex. This process was repeated 3 – 4 times to digest the DMG completely, so that purified Ni could be obtained. A final step for Ni purification was added, by performing another AG1-X8 column to ensure complete Fe removal.
Ni and Zn isotope measurements were carried out by MC-ICP-MS (Neptune Plus, Thermo Scientific) at CRPG-CNRS, University of Lorraine, France. Details on Zn isotope measurements are given in Tang et al. (2012). Briefly, Zn samples were diluted to obtain the same signal as measured in 100 ng g-1 Zn IRMM 3702 solution, and Cu NIST 976 was added to both standard and samples for mass bias correction. In addition to Cu doping, standard-sample-standard correction was carried out to account for the difference between Cu and Zn behavior. The masses measured were 64Zn, 66Zn, 67Zn, 68Zn and 63Cu, 65Cu. Mass dependent fractionation was verified for all samples. Throughout the study, Zn was regularly measured providing a δ66Zn = -0.28 ± 0.05‰ (n=27). Such a value is in agreement with the published values and can be used to recalculate all data against Zn JMC lyon. Meanwhile, reference material BCR-482 (lichen) was digested and analysed, having a δ66Zn = -0.26‰ ± 0.07 (n=3) (Aebischer, pers. comm.), which is in agreement with previous published data (Cloquet et al. 2006).
For Ni isotope measurement, purified Ni was re-dissolved in 1 ml of 0.1 M HNO3. Then the solution was diluted to 150 ng g-1 of Ni before being loaded via the Aridus II (Cetac) into the MC-ICP-MS in medium resolution mode. Spiked-standards NIST 986 and spiked-samples were run at similar concentrations sequentially as in the classic sample-standard bracketing method. The calibration of the double-spike was conducted following the calibration method provided by Rudge et al. (2009). The whole analytical protocol was applied to two reference materials previously characterized, namely BHVO-2 (basalt) and SDO-1(sedimentary rock). Several preparations and measurements of these reference materials showed the values are in agreement with those published (Gall et al. 2012; Gueguen et al. 2013) (60Ni = -0.01 ± 0.05‰ (2SD, n=11) for BHVO-2 and 60Ni = 0.54 ± 0.05‰ (2SD, n=11) for SDO-1).The external reproducibility (2SD) of the method is thus 0.05‰. Ni isotopic compositions are expressed in delta per mill (‰) relative to NIST SRM 986, while Zn composition is relative to IRMM 3702: δ60Ni (‰) = [(60Ni/58Ni)sample / (60Ni/58Ni)NIST 986 – 1] × 1000, δ66Zn (‰) = [(66Zn/64Zn)sample / (66Zn/64Zn)IRMM 3702 – 1] × 1000.
The average isotope compositions of the shoots of T. arvense and A. murale, and of the whole plants were calculated according to the following equations: Ni 60 Zn 66 60 mi ci Nii and 66 mi ci Zni i i Nishoot Znshoot mi ciNi mi ciZn.
Plant biomass and metal concentrations
All the plants grew healthily with the exception of A.m. 50/50, which presented retarded growth symptoms, probably due to Zn toxicity. This is clearly reflected in the plant biomass data (Figure 2.1a). It is noticeable that N. caerulescens achieved similar biomasses in all the three treatments, indicating that the solution Ni and Zn concentrations used in this experiment had no significant effect on its growth.
The Ni and Zn concentrations in plant organs showed clear species-specific patterns (Figure 2.1b, c). T. arvense, the non-hyperaccumulator, took up relatively small amounts of Ni and Zn, with a root-shoot translocation factor (shoot concentration / root concentration) of around 0.1 for both elements. A. murale, the Ni hyperaccumulator, presented high Ni concentrations in shoots (3570 µg/g in A.m. 50/2 treatment), while most of the Zn was sequestrated in roots. N. caerulescens could hyperaccumulate both Zn and Ni in its shoots.
A competition effect between Ni and Zn in the uptake process was also observed. When Zn in solution increased from 2 to 50 µM, the Ni concentrations in shoots of N. caerulescens and A. murale dropped by 38% and 62%, respectively (Figure 2.1b). When Ni in solution increased from 2 to 50 µM, the Zn concentration in N. caerulescens shoots decreased on average by 39%.
Nickel and Zn isotopic compositions.
Figure 2.2 presents the Ni and Zn isotopic compositions (60Ni and 66Zn in ‰) in plants and Figure 2.3a presents the extent of fractionation between plant and solution. All the plants were inclined to absorb light Ni isotopes, with Δ60Niplant-solut ion values ranging from -0.90 to -0.21‰. It is noticeable that the hyperaccumulators had larger isotopic shift (Δ60Niplant-solut ion = -0.90 to -0.63‰), in particular in low Ni treatment (Δ60Niplant -solut ion = -0.90‰ in N.c. 2/2). Compared to Ni however, Zn isotopes had a smaller shift with Δ66Z-nplant -solut ion values of -0.23 to +0.20‰ (Figure 2.3a).
The competition between Ni and Zn also had an influence on the isotopic compositions of both A. murale and N. caerulescens. The Ni isotope fractionation in high Zn treatments (Δ60Niplant-solut ion = -0.11 to -0.07‰) became less pronounced, in comparison with their corresponding low Zn treatments (Δ60Niplant -solut ion = -0.73 to -0.63‰) (Figure 2.3a). This indicated that Zn had a great impact on Ni isotope fractionation during root absorption.
For Zn, shoots were enriched in light isotopes and the isotope fractionation between shoots and roots was relatively large (Δ66Znshoot -root = -0.80 to -0.44‰). By contrast, heavy Ni isotopes were enriched in the shoots of T. arvense (Δ60Nishoot-root = +0.25‰), while the hyperaccumulators A. murale and N. caerulescens still favored light Ni isotopes (-0.47 to -0.14‰), but to a lesser extent relative to Zn (Figure 2.2 and 2.3b).
Ion transport across root cell membrane
Plants generally assimilate metallic nutrients via two pathways, i.e. an apoplastic and a symplastic route. It is presumable that Ni and Zn are taken up mainly through the symplastic pathway, and a purely apoplastic route for the entry into the xylem is of minor significance (Ernst et al. 2002; Kerkeb and Krämer 2003). Thus, uptake of Ni and Zn by plants is mainly controlled by the absorption of root cells and the isotopic signatures of Ni and Zn of the whole plant should represent the uptake mechanisms of root cell membrane.
The effect of high- and low-affinity transport on isotope fractionation could explain what is observed in our experiment. Thlaspi arvense and A. murale, the non- hyperaccumulators of Zn, were isotopically light relative to the solution (-0.23 to -0.10‰) in all the treatments. In contrast, N. caerulescens, the Zn hyperaccumulator, was enriched in heavy isotopes in high Zn treatment (Δ66Znplant-solut ion = +0.20‰). These divergent results suggest that different Zn transport systems are functioning in hyperaccumulators and non-hyperaccumulators. Plants could switch from high- to low-affinity transport systems as the metal concentrations change from deficient to sufficient levels (Epstein and Bloom 2005). Usually, a high-affinity transport system only plays an important role at extremely low concentrations. In bread wheat, 10 nM of Zn2+ is assumed to be the critical concentration between high- and low-affinity transport (Hacisalihoglu et al. 2001). However, high-affinity transport could function in a wider range of concentrations in hyperaccumulators. The first high-affinity transporter gene, ZNT1, has been cloned from N. caerulescens (Pence et al. 2000). This Zn transporter is expressed to very high levels in this hyperaccumulating plant, in both Zn-deficient and Zn-sufficient status. Whereas in the non-hyperaccumulator T. arvense, the transporter is expressed to very low levels in plants grown in Zn-sufficient solution (1 µM). In our case, 2 and 50 µM of Zn were used in the solution culture. Thus for Zn, low-affinity uptake should take effect predominantly in the non-hyperaccumulators T. arvense and A. murale, which resulted in light isotope enrichment. While both high- and low-affinity transport systems were functioning effectively in the hyperaccumulator N. caerulescens, which resulted in a final isotopic shift of +0.20‰ in high Zn treatment.
The Ni isotope fractionation pattern is different from that of Zn. All species presented light Ni isotope enrichment (Figure 2.2, 2.3a), which may reflect the functioning of low-affinity transport systems. This is corroborated with Aschmann et al. (1987) and Redjala et al. (2010), who inferred that Ni is transported through a low-affinity transport system from uptake kinetic studies. Likewise, Assunção et al. (2008) proposed that N. caerulescens seems to express low-affinity systems for Ni accumulation. The hyperaccumulators A. murale and N. caerulescens presented greater isotopic shifts than the non-hyperaccumulator T. arvense in low Zn treatments (-0.90 to -0.63‰ vs. -0.21‰), indicative of a greater permeability for the low-affinity transport systems in hyperaccumulators.
Table of contents :
1. Literature review and objectives of the thesis
1.1 Discovery of metal hyperaccumulators and their application
1.2 Hyperaccumulation mechanisms: general concept
1.3 Ni homeostasis in higher plants: current understanding
1.3.1 Ni uptake process
1.3.2 Xylem loading and transport process
1.3.3 Xylem unloading and leaf comparmentation process
1.3.4 Phloem translocation process
1.4 Objectives of the thesis
2. Isotope fractionation of Ni in hyperaccumulators*
2.2 Materials and Methods
2.2.1 Plant cultivation and harvest
2.2.2 Analytical methods
2.3.1 Plant biomass and metal concentrations
2.3.2 Nickel and Zn isotopic compositions.
2.4.1 Ion chelation in media
2.4.2 Ion transport across root cell membrane
3. Interaction of Ni with other elements in Noccaea caerulescens during the root uptake process
3.2 Materials and methods
3.2.1 Nickel uptake of N. caerulescens under different concentrations of Ni treatments (Expt. 3.1)
3.2.2 Interaction between Ni, Zn, Fe and Co in N. caerulescens (Expt. 3.2)
3.2.3 Gene expression regarding Ni uptake in N. caerulescens (Expt. 3.3)
3.3.1 Biomass, metal uptake and translocation factor of N. caerulescens in Expt. 3.1
3.3.2 Biomass, metal uptake and translocation factor of N. caerulescens in Expt. 3.2
3.4.1 Interaction between Ni and Zn
3.4.2 Interaction between Ni and Fe
3.4.3 Interaction between Ni and Mn, Cu
3.4.4 Interaction between Ni and Co
4. Nickel partitioning in leaves of Noccaea caerulescens during xylem and phloem transport
4.2 Materials and methods
4.2.1 N. caerulescens growing in Ni, Sr and Rb solutions (Expt. 4.1)
4.2.2 Foliar application of 61Ni in N. caerulescens (Expt. 4.2)
4.3.1 Nickel, Rb and Sr accumulation in young and old leaves
4.3.2 Upward and downward movement of Ni in phloem
4.4.1 Nickel partitioning during xylem transport
4.4.2 Nickel partitioning during phloem translocation
4.4.3 Contribution of xylem transport and phloem translocation on Ni accumulation in leaves
5. Nickel speciation in phloem sap of Noccaea caerulescens*
5.2 Materials and methods
5.2.1 Confirmation of the feasibility of the EDTA-stimulated phloem exudation method (Expt. 5.1)
5.2.2 Extraction of phloem exudate from expanding and old leaves of N. caerulescens (Expt. 5.2)
5.3.1 Nickel, Rb and Sr concentrations in Expt. 5.1
5.3.2 Nickel and Zn concentrations in leaves and phloem exudates of expanding and old leaves in Expt.5.2
5.3.3 Organic compounds in phloem exudate of old leaves
5.4.1 Confirmation of the exudate properties
5.4.2 Nickel enrichment in phloem sap
5.4.3 Nickel speciation in phloem sap
6. Main conclusions and future scope
6.1 Main findings of the thesis
6.2 Conceptual model for Ni homeostasis in hyperaccumulators
6.3 Future scope