SUPPORTING INFORMATION Methods S1 & S2, Tables S1–S6, Figs S1–S7 and
Notes S1
Methods S1
Nutrient Solution, Plant Growth, Digestion and Copper Column Purification
The bulk nutrient solution used to grow both plant species consisted of calcium nitrate
(Ca(NO3)2) (2 mM), magnesium sulphate (MgSO4) (0.5 mM), potassium nitrate (KNO3) (1.2
mM), sodium phosphate (NaH2PO4) (0.1 mM), 2-(N-Morpholino)ethanesulfonate (MES) (2.5
mM), sodium chloride (NaCl) (25 µM), boric acid (H3BO3) (15 µM), manganese sulphate
(MnSO4)(5 µM), (NH4)6Mo7O24 (0.07 µM), zinc sulphate (ZnSO4) (1 µM) and CuCl2 (1 µM).
To maintain Fe in a soluble form for plant growth Fe specific chelators were used: N,N'-Bis(2-Hydroxybenzyl) ethylenediamine-N,N'-Dipropionic acid (HBED) for tomatoes and
ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDDHA) for oats. The Fe was added
into the hydroponic solutions as Fe (III)-HBED (12 µM) for the tomato treatments and
Fe(III)-EDDHA (12 µM) for oat treatments. The EDDHA chelate was used for oats, given
the inefficiency of strategy II plants to extract Fe from the HBED complex (Parker &
Norvell, 1999). These chelates were selected based on GEOCHEM modelling of nutrient
solutions at pH 4.5 as they yielded the highest possible “free” Cu(II) concentrations.
Assuming a maximum fractionation during complexation with organic ligands of +0.25‰
(Δ65Cucomplex-free) (Bigalke et al., 2010), with 15% of Cu complexed (85% “free Cu”), the
maximum induced fractionation on the “free Cu” pool is ca. 0.04, which is considered
insignificant.
Seeds of both species were soaked in an aerated solution of 200 µM CaSO4 for two days,
before being transferred to moistened paper towel and germinated in the dark for 7-10 days.
Seedlings were placed in the light for an additional 2 days before 6 tomato and 6 oat seeds of
similar size were transferred to acid washed pots containing 2 l of complete nutrient solution,
with one plant per 2 l pot. Plants were subject to a14-h photoperiod with light intensity of ca.
350 µmol photons m-2 s-1, with day: night temperature 25:15°C, and grown for 30 days.
Sample Digestion
Before plant samples were digested and purified for isotope analysis, the sample preparation
method was tested for Cu digest efficiency and column recovery using the NIST 1573a
(tomato leaves) standard reference material (SRM). Five replicates of NIST 1573a SRM were
digested according to the procedure outlined in the methods, with Cu recovery determined
after digestion (Table S2). Digests were purified according to the method of Marechal et al.
(1999), as outlined in Table S3, and Cu quantitative Cu recoveries confirmed (Table S2). The
recoveries obtained confirmed the digestion and separation methods were effective in
extracting Cu from a plant matrix into solution and that quantitative recovery of Cu could be
achieved. Removal of matrix elements was shown to be effective after three purifications,
with all matrix elements below detection in the Cu fraction (Fig. S1).
After harvest plants ca. 0.05 – 0.1 g of the freeze dried plant tissues (roots, stems, leaves)
were cold digested in 3 ml of nitric acid (HNO3) and 1 ml hydrogen peroxide (H2O2) in
polytetrafluoroethylene (PTFE) vials, followed by a 2 h hotplate reflux at 140°C. Samples
were then evaporated to dryness and redissolved in 7 ml HNO3 and 3 ml H2O2 and transferred
to closed PTFE vessels. The samples were microwave digested in sealed vessels (Ethos E,
Milestone), using the following temperature ramping sequence: 1) temperature ramped to
100°C for 25 min; 2) temperature ramped to 150°C for 25 min, 3) temperature ramped to
180ºC for 25 min, and 4) temperature constant at 180ºC for 15 min. The clear digest solutions
were transferred to PTFE vials, evaporated to dryness and redissolved in 2 ml of 7 M
hydrochloric acid (HCl) for Cu purification. Approximately 0.1 ml of the digested solutions
were taken to determine concentrations of total Cu, major and minor nutrients by inductively
coupled plasma mass spectrometry (ICP-MS) (Agilent 7700) and inductively coupled plasma
– optical emission spectroscopy (ICP-OES) (Spectro ARCOS) (Table S4).
All acids were distilled (DST-1000, Savillex) and dilutions were made with high purity
deionised water (Millipore). All digestions, separations and sample preparations occurred in a
Class 1000 clean room. The total procedural blank for Cu after three column purifications
was 10 ± 5 ng (mean ± SD, n=4), which contributed less than 3% of total Cu analysed. The
post-column Cu yield for the NIST 1573a tomato leaves (n=5) was 102 ± 6% (Table S2), and
for plant samples (roots, stems, leaves) was 100 ± 6%.
Cu isotope ratio analysis
The Cu isotope ratios were expressed as δ65Cu values relative to NIST 976 Cu isotopic
reference material according to Equation 1.
 ( 65 Cu/ 63Cu) sample

δ Cu  1000   65
 1
63
 ( Cu/ Cu) NIST976 
65
Equation 1
Masses 60 (Ni), 61 (Ni), 62 (Ni), 63 (Cu), 64 (Zn + Ni), 65 (Cu), and 66 (Zn) were detected
simultaneously on Faraday cups. NIST 986 Ni standard (62Ni/60Ni = 0.138600) was used for
instrumental mass bias correction of the measured 65Cu/63Cu isotope ratios, using the external
normalisation method, with sample-standard bracketing with the NIST 976 Cu standard
before and after every sample (Ehrlich et al., 2004; Bigalke et al., 2010). The analytical
precision of MC-ICP-MS analysis was determined using an in-house Cu standard prepared
from a digested metallic Cu wire, and used to determine intra-day reproducibility. This wire
standard has a δ65Cu of 0.45 ± 0.04‰ (mean ± 2SD, n = 40), which represents the long-term
reproducibility of Cu isotope measurements.
Table S1. GEOCHEM predicted % distribution of Cu in the three different nutrient solutions
used for plant growth.
Cu complex
% distribution in FeHBED solution
Free Cu2+ ion
PO43SO42NO3OHHBED
EDDHA
94.2
0.25
4.40
1.05
0.04
0.05
-
% distribution in
Fe-EDDHA
solution
88.3
0.22
4.13
0.98
0.04
6.32
% distribution without
Fe
94.2
0.24
4.49
1.05
0.04
-
Table S2. Copper recoveries following digestion and column purification of NIST 1573a
standard reference material.
Sample
% digest recovery
1
2
3
4
5
104
103
95
95
95
% column recovery (3
column purifications
107
92
104
108
103
Table S3. Summary of anion exchange column purification procedure for Cu, using AG –
MP – 1 resin.
Fraction
Sample Load
Matrix
Cu
Fe
Zn
Eluant
7M HCl
7M HCl
7M HCl
2M HCl
0.5M HNO3
Volume (mL)
1
7
35
10
10
120
100
Al
B
% Element Eluted
80
Cu Fraction
Ca
K
Mg
60
Mn
Na
40
P
S
Zn
20
Cu
0
1
6
11
16
21
26
31
36
41
46
51
56
mL of 7M HCl Eluted
Fig. S1. Element elution profiles of NIST 1573a tomato leaf from the anion exchange resin
column using the elution scheme outlined in Table S2.
Table S4. Macro- and micronutrient concentrations (mg kg-1) for tomato and oat plant tissues grown in Fe-sufficient (+Fe) or Fedeficient (-Fe) conditions.
Species
Tomatoes
Oats
Treatment
Plant Tissue
Fe
Ca
Mg
Nutrient (mg kg-1)
P
K
Mn
Zn
+Fe
Root
Leaves
444
125
5070
37648
2549
5757
7737
8524
56986
34072
872
204
287
52
-Fe
Root
Green Leaves
Chlorotic Leaves
64
51
11
4561
57815
25253
3018
8580
6286
8133
10423
10289
38334
21364
28588
1573
401
215
351
74
67
+Fe
Root
Leaves
3902
95
2403
4592
5054
1651
6261
6915
49150
71335
164
188
70
46
-Fe
Root
Green Leaves
Chlorotic Leaves
1034
93
23
2396
6540
2474
3243
1872
1661
5674
4981
8199
49338
68911
62528
178
286
187
100
60
98
Methods S2
XAS Model Compounds and Analysis
Copper speciation in plant tissues was determined using principle component analysis (PCA)
fitting of model compounds of known speciation. XANES Cu K-edge spectra for six Cu
model compounds were collected for this purpose. Copper(II) sulphate (CuSO4), Cu(I)- and
Cu(II)-acetate were all purchased from Sigma Aldrich, and were analysed unaltered. Cu(II)histidine was prepared using CuCl2 (1.7g) and histidine (1.56g), and precipitated in ethanol,
as per the method of Saxena et al. (1996). Cu-cysteine was prepared in a 1:4 mole ratio, from
CuSO4 and L-cysteine hydrochloride, according to the protocol outlined by Dokken et al.
(2009). The method is stated to produce a Cu(II)-cysteine product, however, it was found
during XAS analysis of the model compound that it was not a Cu(II) but a Cu(I) product, and
as such, was treated as a Cu(I) model compound. An aqueous solution of Cu(II)-EDTA
(5mM) was prepared at the beamline, from CuSO4 and EDTA. An aqueous solution of Cu(I)glutathione (GSH) (4mM) was prepared at the beamline from CuSO4 and GSH (Sigma) in a
0.1 M Na3PO4 buffer solution at pH 7, as per the method of Ciriolo et al. (1990). All solid
phase samples were analysed as pellets, diluted with cellulose, loaded on Kapton and snap
frozen in liquid nitrogen before loading into the cryostage. All liquid samples were loaded
into a Lucite sample holder with a clean micro-syringe, and snap frozen in liquid nitrogen
before loading onto the cryostage. The spectra for all Cu standard compounds can be found in
Fig. S2.
XAS data was analysed using the EXAFSPAK software package (George, G.N., SSRL).
PCA fitting indicated that three main components accounted for the majority of the variation
in the sample spectra, and as such, the three model compounds that showed the smallest
target transformation residuals were chosen to fit the data; Cu(II)-histidine, Cu(I)cysteine
and Cu(I)-GSH. The percentage of the spectra that can be attributed to each of the model
compounds can be seen in Table S4, with the resulting linear combination fits for roots and
leaves shown in Figs S3 and S4, respectively. These values were used to derive the mg kg-1
Cu(I) and Cu(II) species present in plant tissues, as presented in Fig. 3.
EXAFS data analysis indicated that the first coordination shell of all samples was dominated
by S ligands, and the fit error on all samples was significantly reduced by adding a second
shell Cu-Cu interaction at ca. 2.7Å (Table S5). The EXAFS spectra, Fourier transformed
spectra and fit spectra for the three root samples analysed are shown in the main text Fig. 5
and Figs S5-6.
Fig. S2. Standard compound Cu K-edge XANES spectra used for principle component
analysis.
Fig. S3. Measured (black solid line) and fitted (purple broken line) XANES spectra of root
samples of tomatoes and oats.
Fig. S4. Measured (black solid line) and fitted (purple broken line) XANES spectra of leaf
samples of tomatoes and oats.
Table S5. Linear Combination fitting of XANES Cu k-edge spectra for tomato and oat plants grown
in Fe sufficient (+Fe) and Fe deficient (-Fe) nutrient solutions.
Cu(I)-Glutathione
Tomato Plants
+Fe
-Fe
Roots
Leaves
Roots
Leaves
58%
43%
69%
45%
Oat Plants
+Fe
-Fe
Roots
54%
Leaves
42%
Roots
49%
Leaves
54%
Cu(I)-cysteine
0%
29%
18%
35%
40%
26%
39%
23%
Cu(II)-histidine
30%
13%
14%
17%
8%
22%
12%
14%
Table S6. Results for EXAFS curve fitting of tomato and oat plant roots. Bold data indicates
the best fit parameters as determined by fit error, physical reasonableness of parameters and
visual inspection of fit spectra. Numbers in italics represent physically unrealistic parameter
values.a
Sample
Tomato –Fe
Shells
1
1
1
2
2
2
Oat +Fe
1
1
1
2
2
2
2
2
2
Oat–Fe
1
1
1
2
2
2
Interaction
Cu-S
Cu-S
Cu-S
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu-S
Cu-S
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu-S
Cu-S
Cu-S
Cu...Cu
Cu-S
Cu...Cu
Cu-S
Cu...Cu
N
2.5
3
3.5
2.5
0.5
3
0.5
3.5
0.5
2.5
3
3.5
2.5
0.5
3
0.5
3.5
0.5
2.5
1
3
1
3.5
1
2.5
3
3.5
2.5
0.5
3
0.5
3.5
0.5
R (Å)
2.250(3)
2.250(3)
2.250(3)
2.2537
2.7073
2.254(3)
2.706(3)
2.256(3)
2.706(3)
2.250(5)
2.250(5)
2.251(5)
2.258(4)
2.688(4)
2.259(4)
2.687(4)
2.259(4)
2.687(4)
2.261(4)
2.686(5)
2.260(4)
2.687(5)
2.260(4)
2.688(4)
2.243(4)
2.247(4)
2.249(4)
2.248(3)
2.652(9)
2.251(3)
2.661(8)
2.253(3)
2.668(7)
σ2
0.0054(2)
0.0069(2)
0.0079(2)
0.00570
0.00251
0.0071(2)
0.0022(2)
0.0081(2)
0.0020(3)
0.0045(3)
0.0056(2)
0.0067(3)
0.0049(3)
0.0011(4)
0.0061(3)
0.0007(3)
0.0072(3)
0.0005(3)
0.0046(2)
0.0051(5)
0.0057(2)
0.0047(4)
0.0068(3)
0.0043(4)
0.0055(2)
0.0067(2)
0.0079(2)
0.0054(2)
0.0054(2)
0.0066(2)
0.0062(8)
0.0078(2)
0.0056(7)
ΔEo (eV)
-14.9(7)
-14.9(6)
-14.8(6)
-14.188
-14.188
-14.2(5)
-14.2(5)
-14.2
-14.2
-15.0(1)
-15.0(1)
-15.0(1)
-14.2(8)
-14.2(8)
-14.2(8)
-14.2(8)
-14.1(7)
-14.1(7)
-13.5(8)
-13.5(8)
-13.6(7)
13.6(7)
-13.6(7)
-13.6(7)
-17.3(8)
-16.6(8)
-16.4(7)
-16.3(7)
-16.3(7)
-15.8(6)
Error
0.456
0.457
0.468
0.355
-15.5(6)
0.428
0.340
0.346
0.580
0.585
0.597
0.492
0.486
0.492
0.511
0.510
0.519
0.463
0.459
0.467
0.426
0.420
The k-range was 1_14.2 Å_1 and a scale factor (S02) of 0.9 was used for all fits. ΔE0 = E0-12 658 (eV) where
E0 is the threshold energy. Values in parentheses are the estimated standard deviation derived from the diagonal
elements of the covariance matrix and are a measure of precision. The fit-error is defined as
[Σk6(χexp -χcalc)2/Σk6χexp2]1/2.
a
Fig. S5. EXAFS spectra (left) and Fourier Transform (right) of oat roots grown in an Fesufficient nutrient solution. Black line = experimental data, green line = fitted data. Fit
parameters can be found in Table S5.
Fig. S6. EXAFS spectra (left) and Fourier Transform (right) of oat roots grown in an Fedeficient nutrient solution. Black line = experimental data, green line = fitted data. Fit
parameters can be found in Table S5.
Notes S1
Rayleigh fractionation model for root-to-shoot translocation
Since the solution was renewed daily to avoid bulk depletion, the δ65Cusolution remained
constant and the δ65Cuwhole plant (relative to the nutrient solution value) indicates the
fractionation in the uptake process, which was around -1‰ for tomato, suggesting a reductive
uptake mechanism. The δ65Cushoot was considerably larger than the δ65Curoot for tomato,
pointing to positive fractionation in the translocation process. The δ65Cushoot and δ65Curoot
values do not only depend on the isotopic fractionation in the translocation process, but also
on the fraction of Cu translocated to the shoot. A Rayleigh fractionation model was used to
estimate the fractionation in the translocation process. Assuming a constant fractionation
factor for the translocation, t, the δ65Curoot value can be calculated as:
δ65Curoot = δ65Cuwhole plant + t .lnf
(S1)
with f the fraction of Cu in the whole plant that is translocated to the shoot. The δ65Cushoot
follows from the mass balance and δ65Cuwhole plant:
δCu 65shoot 
δCu 65 whole plant  (1  f )  δCu 65root
f
(S2)
The fractionation factor, t, was estimated by fitting equation S1 to the measured δ65Curoot for
the tomato plants on both the –Fe and +Fe treatments (Fig. S7). A value of 0.85‰ was
derived, suggesting oxidation of Cu when Cu is loaded in the xylem and translocated to the
shoot.
0.0
root
εt= 0.85‰
shoot
d65Cu
-0.5
whole plant
-1.0
-Fe
-1.5
+Fe
-2.0
0
0.2
0.4
0.6
0.8
1
Fraction Cu translocated to shoot
Fig. S7. The δ65Cu value in root and shoot as function of the fraction Cu translocated to
shoot. Symbols are measured values for tomato on the Fe-sufficient (+Fe) or Fe deficient
(-Fe) treatments (error bars are standard deviations of three replicates). Curves show
predicted values using a Rayleigh fractionation model assuming a constant fractionation
factor during translocation (εt = 0.85‰).